119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep

2020 ◽  
Vol 32 (2) ◽  
pp. 186
Author(s):  
D. Anzalone ◽  
M. Czernik ◽  
L. Palazzese ◽  
Y. Ressaissi ◽  
P. Scapolo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Window ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Basic Research ◽  
Cell Number ◽  
Differential Staining ◽  
Assisted Reproductive Technique ◽  
Significant Difference

The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.

236 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATOR REGULATORS IN ASSOCIATION WITH BMP15 ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 207
Author(s):  
M. F. Machado ◽  
M. F. G. Nogueira ◽  
R. B. Gilchrist ◽  
M. L. Sutton-McDowall ◽  
D. G. Mottershead ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Oocyte Competence ◽  
Significant Difference

BMP15 is a promising peptide to improve oocyte competence; also, addition of cyclic adenosine monophosphate modulator (cAMP) regulators prevents spontaneous maturation in vitro and promotes embryo development. We aimed to assess embryo development after prematuration [pre-in vitro maturation (IVM)] with IBMX and Forskolin (FSK) and maturation in the presence or absence of a purified pro mature region of BMP15. Immature cumulus-oocyte complexes (COC) were cultured in vitroMat (IVF Vet Solutions, Adelaide, Australia) plus 4 mg mL–1 fatty acid free-BSA and rhFSH (0.1 IU mL–1), then divided into the following treatment groups: 1) spontaneous IVM: 24 h of IVM; 2) spontaneous IVM + BMP15: 24 h of IVM in the presence of BMP15 (100 ng mL–1); 3) Pre 2 h: pretreatment with IBMX (500 µM; Sigma-Aldrich) and FSK (100 µM; Sigma-Aldrich) for 2 h following 24 h maturation; and 4) Pre 2 h + BMP15: pretreatment with IBMX and FSK for 2 h following 24 h maturation in the presence of BMP15 (100 ng mL–1). After maturation, oocytes were inseminated and zygotes were cultured for 5 days in VitroCleave (IVF Vet Solutions, Adelaide, Australia) and transferred into VitroBlast (IVF Vet Solutions, Adelaide, Australia) until blastocyst assessment (Days 7 and 8). Zona-intact embryos were retrieved to assess differential staining of trophectoderm and inner cell mass. Data were transformed into a logarithm and analysed by 1-way ANOVA and post hoc least significant difference using SigmaStat software (SPSS Inc., San Jose, CA, USA; P < 0.05). There was no difference among groups on cleavage rates or blastocyst rates at Day 7; however, both Pre 2 h treatments increase hatched blastocyst rates at Day 8 of embryo development (Table 1). Supplementation with BMP15 increased total blastocyst rates at Day 8, regardless of pretreatment with IBMX+FSK (Table 1). Our data demonstrate that embryos from oocytes matured in the presence of BMP15 or pretreated with IBMX+FSK increase trophectoderm and total cell numbers; however, no differences were observed for inner cell mass. We conclude that Pre 2 h treatment or BMP15 increase embryo development; however, no effect of cAMP regulators in association with BMP15 on embryo development was observed. Table 1.Embryo development Supported by FAPESP (project numbers: 2012/1073-8; 2013/12960-9; 2013/05083-1; 2012/50533-2).

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

157 EFFECT OF MELATONIN ON EMBRYO QUALITY OF BOVINE OOCYTES SUBJECTED TO HEAT SHOCK

2016 ◽  
Vol 28 (2) ◽  
pp. 208
Author(s):  
N. G. Alves ◽  
I. J. Ascari ◽  
L. S. A. Camargo ◽  
J. Jasmin ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
Embryo Quality ◽  
Linear Models ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Quality Data ◽  
Trophoblast Cells ◽  
Melatonin Concentration

The aim of this study was to evaluate the effect of different concentrations of melatonin added to in vitro maturation (IVM) medium of oocytes subjected to heat shock on embryo quality. Immature oocytes aspirated from ovaries obtained from a slaughterhouse were selected and randomly allocated in factorial experiment design (3 × 2). Three concentrations of melatonin (0, 10–6, and 10–4 M; M5250, Sigma, St. Louis, MO, USA) were added to the IVM medium and 2 incubation conditions (conventional: 24 h at 38.5°C and 5% CO2; heat shock: 12 h at 41°C followed by 12 h at 38.5°C and 5% CO2) were tested, resulting in treatments: M1 (0 M; 38.5°C; n = 15), M2 (10–6 M; 38.5°C; n = 15), M3 (10–4 M; 38.5°C; n = 15), M4 (0 M; 41°C; n = 15), M5 (10–6 M; 41°C; n = 15), and M6 (10–4 M; 41°C; n = 15). The IVM was performed in a Nunc plate (144444 – Thermo, Fisher Scientific Inc., Pittsburgh, PA, USA) containing 400 µL of TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20 µg mL–1 of FSH (Pluset®, Calier Laboratories, Barcelona, Spain) and 10% oestrus cow serum. Oocytes were IVF in FERT-TALP medium for 20–22 h and incubated at 38.5°C and 5% CO2. After IVF, the presumptive zygotes were denuded and cultured in CR2aa medium supplemented with 2.5% FCS (Nutricell, Campinas, Brazil) in an incubator at 38.5°C under 5% CO2, 5% O2, and 90% N2, and saturated humidity for 8 days. Blastocysts with 8 days post-fertilization from different treatments were fixed in 4% paraformaldehyde in PBS for 1 h and analysed by TUNEL assay (Deadend™ Fluorometric TUNEL System, Promega, Madison, WI, USA) to evaluate embryonic quality. Data were analysed by generalised linear models considering the Poisson distribution and using the Proc Genmod of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA) considering effects of melatonin concentration, incubation conditions, and interaction between the factors. Values shown are the mean ± s.e.m. The interaction between melatonin concentration and incubation conditions was no significant (P > 0.05). The total number of cells was not affected (P > 0.05) by melatonin, but it was decreased (P < 0.05) by heat shock (M1 = 117 ± 6.7a; M2 = 118 ± 4.2a; M3 = 120 ± 6.3a; M4 = 102 ± 6.2b; M5 = 106 ± 5.7b; M6 = 108 ± 8.9b). Melatonin and heat shock did not affect (P > 0.05) the index of embryo apoptotic cells (M1 = 15.3% ± 2.0; M2 = 15.5% ± 1.3; M3 = 13.6% ± 2.0; M4 = 14.9% ± 1.5; M5 = 13.3% ± 1.3; M6 = 13.5% ± 1.2) and the index of trophoblast cells (M1 = 74.6% ± 2.3; M2 = 75.0% ± 1.7; M3 = 75.2% ± 1.9; M4 = 78.4% ± 2.3; M5 = 76.4% ± 3.0; M6 = 75.2% ± 2.6). The melatonin and heat shock affected the index of the inner cell mass (ICM; P < 0.05), and the heat shock reduced the index of the ICM of oocytes not treated with melatonin (M1 = 25.4% ± 2.3a; M2 = 25.0% ± 1.7a; M3 = 24.8% ± 1.8a; M4 = 21.6% ± 2.3b; M5 = 23.6% ± 3.0a; M6 = 24.8% ± 2.6a). In conclusion, melatonin supplementation to the medium IVM of oocytes subjected to heat shock had no effect on blastocyst total cell number, general apoptotic index, or index of the trophoblast cells, but increased index of the ICM. Research was supported by Fapemig, CNPq, Embrapa, and CAPES.

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 129
Author(s):  
G.-S. Im ◽  
J.-S. Seo ◽  
I.-S. Hwang ◽  
S.-W. Kim ◽  
H.-S. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Electric Pulse ◽  
Chemical Activation ◽  
Cell Mass ◽  
Cell Number ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

182 FAST STAINING METHODS FOR DIFFERENTIAL STAINING OF INNER CELL MASS AND TROPHECTODERM CELLS OF MAMMALIAN BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 199
Author(s):  
S. W. Kim ◽  
J.-K. Park ◽  
J.-H. Han ◽  
C. G. Park ◽  
W.-K. Chang
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Treatment Method ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Green Color ◽  
Mixture Treatment

The present study was undertaken to develop a simple differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using the permeabilizing agent, saponin, without species-specific antibodies and complements. The nuclei of whole embryos were pre-stained to green by 5 �M SYTO 13 for 10 min. After washing, the green color of TE was turned to red by exposure to 100 �g/mL propidium iodide and 50 to 100 �g/mL saponin solution for 5 to 10 min. To confirm the exactness of staining patterns, the fluorescent nuclei of ICM and TE from mouse, pig, and bovine blastocysts were compared with 3D location by confocal microscopy. By the saponin mixture treatment method, in vitro-cultured mouse, pig, and bovine blastocysts were shown to have an ICM:TE ratio of 1:2.5, 1:4.5, and 1:3.6, with an average total cell number of 78 � 14 (n = 45), 65 � 18 (n = 49), and 150 � 20 (n = 45), respectively. Although a few TE cells were stained to a yellowish-green color, the successful protection of the green color of ICM depended on the exposure time of blastocysts to the saponin mixture. The total time lapse of the procedure did not exceed 1 h. These results indicate that saponin could be used as a practical substitute for special antibodies and complements. So this differential staining for examining the ICM:TE ratio and the total cell count of mammalian blastocysts would be a fast and reliable method.

scholarly journals Stage specific response in early mouse embryos exposed to prednisolone in vitro

2020 ◽  
Author(s):  
Shubhashree Uppangala ◽  
Akshatha Daddangadi ◽  
Jeena Susan Joseph ◽  
Sujit Raj Salian ◽  
Riddhi Kirit Pandya ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Recurrent Miscarriage ◽  
Cell Mass ◽  
Cell Number ◽  
Early Embryo ◽  
Specific Response ◽  
Clinical Value ◽  
Developmental Potential

Corticosteroids are increasingly being used during the peri-implantation period to treat women with repeated IVF failure and recurrent miscarriage. However, the direct effects of prednisolone (PRDL), one of the commonly used corticosteroids on early embryo development is not understood. To mimic the possible clinical scenario and to understand the embryonic response to direct PRDL exposure, this pilot study was conducted in a mouse model. Cleavage stage embryos exposed to 3 and 30µM PRDL in vitro were assessed for peri-implantation developmental potential, genetic integrity, inner cell mass (ICM) proliferation and pluripotency markers in the proliferated ICM cells. Exposure to 30µM PRDL delayed the embryonic progression beyond compaction (P<0.05) in comparison to vehicle control and, had reduced total cell number (P<0.001) than all other groups. In addition, 30µM PRDL exposure resulted in poor hatching potential (P<0.05) and increased apoptosis in blastocysts (P<0.05) compared to 3µM PRDL. On the other hand, completely formed ICM outgrowths were significantly higher (P<0.05) in 3µM PRDL compared to control. However, no significant differences were observed in the expression of pluripotency genes. In conclusion, the trend observed in embryos exposed to PRDL in vitro provides important information concerning the use of this drug when treating patients at the peri-implantation phase of IVF cycles. However, the clinical value of this observation on human embryo development needs further research.

131 EFFECTS OF HIGH MOLECULAR WEIGHT HYALURONAN ON SHEEP EMBRYO DEVELOPMENT AND SURVIVAL AFTER CRYOPRESERVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 213
Author(s):  
V. Ghaffarilaleh ◽  
F. Ghafari ◽  
M. Teresa-Paramio ◽  
A. Fouladi-Nashta
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Apoptotic Cell ◽  
Cell Mass ◽  
Differential Staining ◽  
Embryo Viability ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Large Size

Hyaluronan (HA), a component of extracellular matrix in mammalian tissues including that of the reproductive system, has been shown to support embryo development. HA is produced in various sizes with distinct physiological functions. Cleaved sheep embryos produced after in vitro maturation and in vitro fertilization were cultured in serum free synthetic oviduct fluid medium supplemented with increasing concentrations (0, 0.25, 0.5, and 1 mg mL–1) of large size HA (Healon; 6 × 106 Da). Development to blastocyst stage was recorded at Day 7 when a group of the embryos were fixed and stained by differential staining combined with TUNEL labelling to analyse embryo quality. The remainder of the blastocysts from each treatment/repeat were vitrified in open pulled straws and then cultured for an extra period of 48 h to analyse their survival rate and quality after cryopreservation. SPSS version 20 software (SPSS Inc., Chicago, IL, USA) was used for analyzing the data with generalized linear model. Healon did not change blastocyst (33 ± 5.7, 32 ± 6.0, 35 ± 5.5; P ≥ 0.05) or survival rates (63 ± 17.1, 83 ± 15.2, 58 ± 14.2; P ≥ 0.05) as compared to the respective controls (25 ± 5.2, 38 ± 17.1). It increased the total cell (TC) number (83.6 ± 4.6, 100.7 ± 3.8, 97.2 ± 3.7, 105.0 ± 3.9; P ≤ 0.05) and trophectoderm cells (TE) (58.4 ± 3.8, 74.2 ± 3.2, 75.6 ± 3.3, 80.1 ± 3.4; P ≤ 0.05) but had no effect on the number of inner cell mass (ICM) and apoptotic cells. The ICM : TE ratio was not affected (0.45 ± 0.04, 0.36 ± 0.03, 0.33 ± 0.03, 0.30 ± 0.03; P ≥ 0.05). Surviving embryos had higher TC (63.2 ± 3.7, 130.8 ± 3.6, 113.9 ± 5.2, 149.8 ± 5.4; P ≤ 0.05), TE (42.9 ± 3.0, 96.7 ± 3.1, 85.2 ± 4.5, 111.9 ± 4.7; P ≤ 0.05) and ICM (20.3 ± 2.2, 32.9 ± 1.8, 27.7 ± 2.6, 36.5 ± 2.7; P ≤ 0.05). The apoptotic cell numbers and ICM : TE ratio of the survived embryos after cryopreservation were not affected by HA supplementation. The results indicate that large size HA improves the embryo viability and quality, which may have implication for improving embryo transfer.

70 Energy metabolites induce differential DNA methylation levels and transcription in trophectoderm and inner cell mass

2021 ◽  
Vol 33 (2) ◽  
pp. 142
Author(s):  
J. Ispada ◽  
C. B. de Lima ◽  
E. C. dos Santos ◽  
A. M. da Fonseca Junior ◽  
J. V. Alcantara da Silva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Significant Difference

DNA methylation/demethylation is one of several epigenetic mechanisms by which metabolism regulates gene expression. More specifically, α-ketoglutarate (αKG) and succinate (Suc) are tricarboxylic acid cycle metabolites that may decrease and increase, respectively, the activity of DNA demethylases. Because pre-implantation embryos undergo reprogramming in both DNA methylation and metabolic pathways, it is possible that metabolic changes influence this epigenetic mark. To test that hypothesis, bovine embryos were invitro produced by using standard protocols and, 8h after fertilization, zygotes were transferred to synthetic oviductal fluid (SOF)-based culture medium (control, CO) or culture medium containing 4mM dimethyl-αKG, or 4mM dimethyl-Suc, where they remained until Day 4. Embryos were collected at Day 4 or remained in culture until Day 7, in control medium. Day 4 embryos were evaluated for DNA methylation levels by immunofluorescence detection of 5-methylcytosine (5mC) and cleavage rate. Day 7 embryos were also assessed for DNA methylation by immunofluorescence of 5mC, total cell number, blastocyst rates, and quantification of ACTB (housekeeping), DNMT1, DNMT3A, and DNMT3B transcript by RT-qPCR in trophectoderm (TE) and inner cell mass (ICM) separated by immunosurgery. The mRNA expression levels of were normalized to internal control ACTB and subsequently calculated using the 2−ΔΔCT method, using the control group for comparisons. All data were submitted to outlier detection using ROUT with Q=1% followed by one-way analysis of variance (ANOVA) and Fisher’s least significant difference (l.s.d.) test in GraphPad Prism. αKG and Suc did not influence cleavage or blastocyst rates, total cell number, or cell allocation. αKG supplementation reduced 5mC fluorescence intensity in embryos assessed at Day 4 (CO: 12.8±0.4 AU; αKG: 9.0±0.2AU; P&lt;0.0001) and Day 7 (CO: 36.5±0.7 AU; αKG: 23.5±0.4 AU; P&lt;0.0001), whereas Suc incubation increased DNA methylation levels in embryos at Day 4 (CO: 12.8±0.4 AU; Suc: 15.7±0.3 AU; P&lt;0.0001) and Day 7 (CO: 36.5±0.7 AU; Suc: 70.5±0.5 AU; P&lt;0.0001). αKG increased expression of DNMT1 (P=0.0438) in the ICM and led to lower levels of DNMT1 (P&lt;0.0001), DNMT3A (P=0.0013), and DNMT3B (P=0.0015) in TE cells. The culture with Suc increased DNMT1 (P=0.0074), DNMT3A (P=0.0186), and DNMT3B (P=0.0286) in ICM. Regarding TE, Suc resulted in lower expression of DNMT1 (P&lt;0.0001), DNMT3A (P=0.0017), and DNMT3B (P=0.0052). In conclusion, both supplementations resulted in global DNA methylation changes without affecting embryo development rates or morphology. These changes were accompanied by alterations in transcript profiles between ICM and TE, with differences among treatments being more pronounced in transcripts from ICM. This is the first report of DNA demethylation–induced changes by analogues of TCA cycle metabolites during early reprogramming of the bovine embryo with prolonged effects in TE and ICM cells. This research was funded by FAPESP: 2017/18384-0; 2018/11668-6.

scholarly journals 299 IN VITRO DEVELOPMENT OF IMMATURE PORCINE OOCYTES FERTILIZED IN VITRO TO THE BLASTOCYST STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 300
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
S.Y. Medvedev ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Significant Difference

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in this study. After in vitro maturation (IVM) for 48 h of cumulus-oocyte complexes, 75.4% (n = 442) of them extruded a visible polar body (PB). Most oocytes with a polar body (PB+ group) were found to be at metaphase II (M-II) stage (91.4%). Most oocytes without a visible polar body (PB− group, n = 144) appeared to be arrested at the germinal vesicle (GV) (41.6%) and first meiotic metaphase (M-I) (34.0%) stages. After IVF of oocytes (the day of IVF = Day 0), there was no significant difference between PB+ and PB− groups in rates of sperm penetration, monospermy, and oocyte activation after the penetration. Embryonic development was assessed by staining with 1% orcein. On Day 2, although there was no difference between the embryo cleavage in PB+ (n = 447) and PB− (n = 217) groups (47.0% and 35.9%, respectively), PB+ embryos had more cells than the PB− embryos (3.37 and 2.81 cells, respectively) (P < 0.05; ANOVA). On Day 4, the cleavage rate of PB+ embryos was higher than that of PB− embryos (45.4% and 24.3%, respectively), and PB+ embryos had more cells than the PB− embryos (8.26 and 6.0 cells, respectively) (P < 0.05; ANOVA). On Day 6, a significantly higher number of PB+ embryos developed to the blastocyst stage than that of the PB− embryos (34.6% and 20.7%, respectively) (P < 0.05). However, by subtracting the GV oocytes from the PB− group, there was no difference in blastocyst rates between the M-I arrested and M-II oocytes (35.3% and 34.6%, respectively). The number of blastomer nuclei in embryos obtained from the PB+ group (52.0) was significantly higher than that of the PB− group (29.1); however, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ significantly (1:1.9 and 1:2.2, respectively) (P < 0.05). Chromosome analysis revealed that PB+ blastocysts had significantly more diploid blastomeres (69.7%) than PB− blastocysts (44.0%), whereas PB− blastocysts had significantly more triploid cells (34.0%) compared with PB+ oocytes (8.4%)(P < 0.05; χ2 test). These results indicate that porcine oocytes arrested at the M-I stage undergo cytoplasmic maturation during culture and have the same ability to develop to blastocysts after IVF as M-II oocytes but with a lower cell number; the latter might be caused by the slower embryonic development.

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