23 Biopsied invitro-produced bovine blastocysts survive vitrification better than slow freezing

2021 ◽  
Vol 33 (2) ◽  
pp. 119
Author(s):  
V. Najafzadeh ◽  
J. Secher ◽  
A. Andersen ◽  
N. Jørgensen ◽  
L. Strøbech ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Slow Freezing ◽  
Cell Counting ◽  
Expansion Rate ◽  
Tunel Staining ◽  
Dead Cell ◽  
Average Cell ◽  
Cell Numbers ◽  
Cell Counts

Trophectoderm (TE) biopsying for single nucleotide polymorphisms (SNPs) analysis is being implemented as a tool for the selection of elite bovine embryos. This biopsy method renders ample cells for analysis without compromising the inner cell mass (ICM), and the blastocyst recovers quickly after biopsy. To use the SNP data for embryo selection before the transfer, the blastocysts need to be cryopreserved either with vitrification or slow-freezing. In intact invitro-produced (IVP) blastocysts, vitrification has already proven optimal regarding embryo survival and pregnancy rates. This study aimed to investigate which cryopreservation approach is superior regarding blastocyst re-expansion rate as well as ICM, TE, and dead cell numbers after biopsying bovine IVP blastocysts. All IVP media and vitrification kits were from IVF Bioscience, and ethylene glycol with sucrose was from EggTech. Oocytes from slaughterhouse ovaries were used for blastocyst IVP. At Day 7, the blastocysts were pooled and randomised into 3 groups: (1) biopsy-control (BC), (2) biopsy-vitrification (BV), (3) biopsy-slow freeze (BSF). Subsequently, 5 to 10 TE cells were biopsied in BO-transfer medium using a 25-µm (inner diameter) biopsy pipette and flicking against the holding pipette. The BC group was incubated at 6% CO2 and 38.8°C for 5h. After scoring the re-expansion rate, the blastocysts were fixed with 4% paraformaldehyde/sucrose for further analyses. The BV and BSF groups were subjected to cryopreservation/thawing protocols according to the manufacturers’ instructions. Both groups, recovered under the same culture conditions as BC, were subsequently scored for re-expansion rate and finally fixed. For cell counting, the embryos were stained with Hoechst (DNA) and CDX2 (TE), combined with the TUNEL staining. ImageJ software (National Institutes for Health) was used for cell counting. P<0.05 was considered statistically significant and was determined using Fisher’s exact test for blastocyst re-expansion rate and Student’s t-test for cell numbers. The re-expansion rate in BV was 81% (61/75), which was significantly lower than in BC (95%; 225/236; P<0.005). In BSF, the re-expansion rate was 52% (28/54), which was significantly lower than in both BV and BC (both P<0.005). For cell counts, 18, 18, and 14 embryos, pooled from 3 to 4 independent IVP replicates, were analysed in BC, BV, and BSF, respectively. The average numbers of ICM cells in BV and BSF were 36±5 and 37±11, respectively, and neither was statistically different from BC (34±7; P>0.05). The average cell counts for TE cells in BV and BSF were 59±6 and 47±10, respectively, and neither was statistically different from BC (56±8; P>0.05). The average numbers of dead cells in BV and BSF were 8±3 and 9±2, respectively, and neither was statistically different from BC (10±4; P>0.05). In summary, the biopsied bovine IVP blastocysts recovered better after vitrification, and neither of cryopreservation methods had any effect on the numbers of ICM, TE, or dead cells. This project was supported by Innovation Fund Denmark and the Danish Milk Levy Foundation.

81 JAK-STAT SIGNALLING IS CRITICAL FOR INNER CELL MASS DEVELOPMENT IN BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 133 ◽  
Author(s):  
F. Meng ◽  
B. Forrester-Gauntlett ◽  
H. Henderson ◽  
B. Oback
Keyword(s):  
Gene Expression ◽  
Adenylate Cyclase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Development ◽  
Chemical Screening ◽  
Cell Numbers ◽  
Cell Counts ◽  
Generalised Linear Mixed Model ◽  
Or Gene

The inner cell mass (ICM) of mammalian blastocysts comprises 2 transient lineages, namely hypoblast and epiblast, which develop into extra-embryonic and embryonic tissues, respectively. In the mouse, epiblast cells autocrinally secrete fibroblast growth factor (FGF) to induce hypoblast differentiation, and pharmacological FGF/mitogen-activated protein kinase (MAPK) signal inhibition converts all ICM cells into epiblast. We conducted a chemical screen for additional signal enhancers of epiblast identity in bovine Day 8 blastocysts. From the morula stage onwards, in vitro-fertilised (IVF) embryos were cultured in the presence of 9 small molecule inhibitors, targeting 9 principal signal pathway components. Inhibitors included SB431542, LDN193189, BIBF1120, Forskolin, BI-D1870, A66/TGX 221/ZSTK474, and AZD1480, targeting TGFβ-RI, BMP-RI, VEGFR/PDGFR/FGFR, adenylate cyclase, ribosomal S6 kinase (RSK), PI3K, and JAK2 signalling, respectively. Using (1) blastocyst quality (by morphological grading), (2) cell numbers (by differential stain), and (3) lineage-specific candidate gene expression (by quantitative PCR) as readouts, we sought to identify positive and negative regulators of ICM development and lineage determination. Based on our previous digital mRNA profiling data (McLean et al. 2014 Biol. Reprod., in press), we selected discriminatory epiblast-specific (FGF4, NANOG) and hypoblast-specific (PDGFRα, SOX17) markers for qPCR analysis. Each inhibitor was compared, alone or in combination, to an appropriately diluted dimethylsulfoxide (DMSO) vehicle control in at least 3 biological replicates. Statistical significance was determined using a generalised linear mixed model with binomial distribution and logit link for developmental data and REML for log cell counts and log gene expression data, applying fixed treatment effects and random run and sample within run effects. Blocking TGFβ1-, BMP- or VEGF-/PDGF-/FGF-signalling did not affect blastocyst development, ICM v. trophectoderm (TE) cell numbers, or gene expression. Repression of PI3K signals via AG66 and TGX, but not ZSTK alone, modestly decreased grade 1–2 blastocyst development (P < 0.05) but had no effect on cell numbers or gene expression. Stimulating adenylate cyclase activity increased NANOG levels (2.5-fold; P < 0.05), while RSK inhibition reduced FGF4 and PDGFRα expression (4-fold and 2-fold, respectively; P < 0.05). Suppressing JAK-STAT signalling, on the other hand, consistently compromised grade 1–2 blastocyst development and ICM numbers relative to DMSO controls (18/235 = 7% v. 59/159 = 29%, n = 5 IVF runs; 12 v. 47 ICM cells, N = 25 and N = 7 embryos counted, respectively; P < 0.0001). Epiblast and hypoblast markers were up to 40-fold reduced (FGF4, NANOG, SOX17; P < 0.0001) or completely abolished (PDGFRα; P < 0.0001). This effect was specific to the ICM because TE numbers and TE-specific gene expression (CDX2, KTR8) were not significantly altered. In summary, we have established Day 8 blastocysts as a useful chemical screening platform and demonstrated that bovine ICM development critically depends on JAK-STAT signalling.

113 RETINOIC ACID DOES NOT PROTECT AGAINST THE SELECTIVE DAMAGE TO THE BOVINE INNER CELL MASS INFLICTED BY VITRIFICATION

2007 ◽  
Vol 19 (1) ◽  
pp. 174
Author(s):  
C. Díez ◽  
A. Rodríguez ◽  
C. De Frutos ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Counting ◽  
Bovine Embryo ◽  
Embryo Survival ◽  
Binding Effect ◽  
Cell Counts ◽  
Before And After

Successful cryopreservation of in vitro-produced embryos is a major objective in reproductive biotechnology. It was reported that in vitro culture with high BSA concentrations improved bovine embryo survival after vitrification (D�ez et al. 2005 Reprod. Dom. Anim. 40, 384). All-trans retinoic acid (ATRA) increases cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodr�guez et al. 2006 Hum. Reprod. 21, 2149–2157). This work analyzed the effect of ATRA on bovine embryo development, survival to vitrification, and cell allocation before and after cryopreservation. Bovine cumulus–oocyte complexes were matured and fertilized in vitro, and presumptive zygotes cultured in SOF + 20 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 917 morulae + early blastocysts were cultured for 24 h with: (1) 1.4 �M ATRA, (2) 0.7 �M ATRA, and (3) no ATRA (control). Embryos were subsequently cultured up to Day 9 in SOF + 20 g L-1 BSA. Development was recorded and differential cell counting was performed on Day 8 and 9 hatched blastocysts. Simultaneously, Day 7 and 8 expanded blastocysts were vitrified (OPS; Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). After warming, blastocysts were cultured for 72 h in B2 + 5% FCS with Vero cells, and cell counts were performed in fully expanded or hatched blastocysts. Data (7 replicates for cell counts before and 4 after vitrification) were processed by GLM and Duncan&apos;s test, and were expressed as LSM � SE (x,y: P = 0.01; a,b: P &lt; 0.05; α,β: P &lt; 0.002). Developmental rates did not differ among groups. Blastocysts cultured in 0.7 �M ATRA survived vitrification at rates similar to those of controls, and only hatching rates 24 h post-warming were significantly lower than those of controls (4.0 � 8.2a vs. 31.2 � 8.2b). ATRA at 1.4 �M was detrimental to survival of Day 7 embryos, whereas differences were not detected in Day 8 blastocysts. In all groups, the vitrification procedure significantly reduced the cells of the ICM (1.4 �M ATRA: 28.3 � 3.1α vs. 8.6 � 4.1β; 0.7 �M ATRA: 27.7 � 3.5α vs. 2.2 � 4.1β; Control: 31.3 � 3.1α vs. 7.0 � 5.1β). Total cell counts were: 1.4 �M ATRA: 160.0 � 9.8a vs. 130.0 � 12.2b; 0.7 �M ATRA: 165.3 � 8.8a vs. 123.2 � 11.7b; Control: 161.2 � 9.2a vs. 131.0 � 15.1b. The ratios of ICM/TE cells were: 1.4 �M ATRA: 16.9 � 2.7x vs. 6.1 � 3.2y; 0.7 �M ATRA: 17.2 � 2.3x vs. 2.0 � 3.0y; Control: 20.6 � 2.4x vs. 4.3 � 3.9y. All values are before and after vitrification, respectively. When considered together, the differences in the cell counts before and after vitrification were highly significant (*P &lt; 0.0001): 1.4 �M ATRA: 29.2 � 1.9* vs. 5.9 � 2.6; 0.7 �M ATRA: 162.5 � 5.5* vs. 127.2 � 7.6; Control: 18.3 � 1.5* vs. 4.2 � 2.0. Our results show that ATRA did not improve the embryo survival to vitrification. Although 1.4 �M ATRA was used to avoid a 'binding effect' related to an elevated protein level (Klaassen et al. 1999 Biochim. Biophys. Acta 1427, 265–275), the BSA concentrations used in culture could mask any ATRA effect. The vitrification procedure used in this study produced a selective damage within the ICM cells, which can explain the reduced survival rates obtained after warming. This work was supported by Grant AGL2005-04479.

127 BINDING RETINOID RECEPTORS BY SPECIFIC AGONISTS AFFECTS THE BOVINE BLASTOCYST DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 172 ◽  
Author(s):  
E. Gómez ◽  
A. Rodríguez ◽  
C. Alonso-Montes ◽  
N. Caamaño ◽  
L. J. Royo ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Inner Cell Mass ◽  
Retinoic Acid Receptor ◽  
Cell Mass ◽  
Cell Counting ◽  
Retinoid Receptor ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Development And Differentiation

Production of embryos in vitro with improved inner cell mass (ICM) and high ICM per total cell rate is a major objective in reproductive biotechnology. Exogenous all-trans retinoic acid (ATRA), a vitamin A metabolite, and endogenous retinoid regulate development and differentiation during bovine morula to blastocyst transition in vitro. ATRA binds to retinoic acid-receptor (RAR), and the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). The unspecific binding of 9-cis-RA to receptors makes it difficult to study RXR transactivation. Therefore, in this work we studied blastocyst development and cell counts by using a specific synthetic RXR agonist [LG100268 LG; a gift of Ligand Laboratories] as opossed to the effect exerted by ATRA upon RAR binding. Cumulus-oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in B2 medium with Vero cells until 139 h post-insemination (Day 6), the time at which embryos [morulae (e90%) + early blastocysts] underwent treatments for 48 h in 400 �L of SOFaaci + 5% FCS. Data (5 replicates per experiment) were analyzed by CATMOD for effects, processed by GLM and Duncan's test, and expressed as LSM � SE (a,b,c P d 0.05). After a LG dose-response experiment (n = 480 morulae), blastocysts rates from LG 1 �M on Day 7 were higher than LG 10 �M, LG 0.1 �M, and LG 0 �M (Day 7: 42.8 � 4.1 vs. 34.4 � 3.7, 36.8 � 3.7, and 32.4 � 3.7, respectively). On Day 8, LG 1 �M also yielded more blastocysts than LG 0.1 �M (50 � 4.2 vs. 44.4 � 3.7, respectively). By differential cell counting (n = 113 blastocysts), hatched blastocysts with LG 10 �M showed proliferation in the ICM, while trophectoderm (TE) cells decreased conversely to LG concentration. These effects were not obvious in expanded blastocysts. In a subsequent experiment (n = 340 morulae), ATRA led to blastocysts rates on Day 8 that were higher than negative, untreated controls, but not different from LG 1 �M (42.4 � 2.4 vs. 33.1 � 2.0 and 36.0 � 2.4, respectively). ATRA and LG 1 increased TE in expanded blastocysts (n = 42) (102 � 13.2 and 96.23 � 13.2, respectively vs. 72.8 � 10.9 in the untreated group) but not in their hatched counterparts (n = 44). There were no differences in the ICM; but percentages of ICM per total cells were higher in hatched blastocysts cultured with ATRA than in expanded LG 1 �M blastocysts and expanded controls (39.5 � 5.5 vs. 24.2 � 5.7, and 20.9 � 4.7, respectively). Manipulation of retinoid receptor-specific pathways make it possible to control blastocyst development and differentiation, leading to embryos of improved quality and viability. Work is in progress to analyze gene expression in these blastocysts. This work was supported by grant MCYT, project AGL-2005-04479.

scholarly journals Cell numbers and cell sizes in organs of mice selected for large and small body size

1978 ◽  
Vol 31 (3) ◽  
pp. 287-301 ◽  
Author(s):  
D. S. Falconer ◽  
I. K. Gauld ◽  
R. C. Roberts
Keyword(s):  
Small Body ◽  
Cell Mass ◽  
Strain Differences ◽  
Cell Number ◽  
Organ Weight ◽  
Cellular Growth ◽  
Average Cell ◽  
Cell Numbers ◽  
Cell Counts ◽  
Liver And Kidney

SUMMARYCell numbers in four organs of large, control and small mice were estimated by nuclear counts. Average cell mass was estimated from the cell number and the organ weight. The mice were from the selected Q-strain with six replicate lines in each size-group. The organs were lung, liver, spleen and kidney. At 6 weeks of age the large mice had more cells and larger cells than the controls in all organs; the small mice had fewer and smaller cells than the controls. The regression of log cell-number on log-organ weight provides a measure of how much, proportionately, cell number contributes to the differences in organ weight. In the lung and spleen, cell number contributed about 70% of the strain differences in organ weight, cell mass contributing about 30%; in the liver and kidney the relative contributions were about equal, at 50%.Cell counts at different ages from 3 to 15 weeks showed that cell number and cell mass contributed to the increases of organ weights during growth in roughly the same proportions as stated above. From this it is concluded that the main effect of selection for body weight has been to speed up or slow down the normal processes of cellular growth.

scholarly journals Interleukin-6 promotes primitive endoderm development in bovine blastocysts

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Interleukin 6 ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Janus Kinase ◽  
Primitive Endoderm ◽  
Cell Numbers ◽  
Downstream Target ◽  
Dependent Signal ◽  
Endoderm Development

Abstract Background Interleukin-6 (IL6) was recently identified as an embryotrophic factor in bovine embryos, where it acts primarily to mediate inner cell mass (ICM) size. This work explored whether IL6 affects epiblast (EPI) and primitive endoderm (PE) development, the two embryonic lineages generated from the ICM after its formation. Nuclear markers for EPI (NANOG) and PE (GATA6) were used to differentiate the two cell types. Results Increases (P < 0.05) in total ICM cell numbers and PE cell numbers were detected in bovine blastocysts at day 8 and 9 post-fertilization after exposure to 100 ng/ml recombinant bovine IL6. Also, IL6 increased (P < 0.05) the number of undifferentiated ICM cells (cells containing both PE and EPI markers). The effects of IL6 on EPI cell numbers were inconsistent. Studies were also completed to explore the importance of Janus kinase 2 (JAK2)-dependent signaling in bovine PE cells. Definitive activation of STAT3, a downstream target for JAK2, was observed in PE cells. Also, pharmacological inhibition of JAK2 decreased (P < 0.05) PE cell numbers. Conclusions To conclude, IL6 manipulates ICM development after EPI/PE cell fates are established. The PE cells are the target for IL6, where a JAK-dependent signal is used to regulate PE numbers.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

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.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 137
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
D. Hermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Cell Numbers ◽  
Cloning Method

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos. Table 1.Cell number and H3K18ac level1

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
S. M. Bernal-Ulloa ◽  
A. Lucas-Hahn ◽  
P. Aldag ◽  
D. Herrmann ◽  
U. Baulain ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Differential Staining ◽  
Final Phase ◽  
Cell Numbers ◽  
Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

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