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.

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

53 TARGETED SCREEN FOR AMINO ACIDS THAT REGULATE BOVINE INNER CELL MASS DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 156
Author(s):  
V. Najafzadeh ◽  
R. Martinus ◽  
B. Oback
Keyword(s):  
Inner Cell Mass ◽  
Statistical Significance ◽  
Cell Mass ◽  
Positive Control ◽  
Drop Out ◽  
Blastocyst Development ◽  
Acetyl Coa ◽  
Cell Numbers ◽  
Fetal Fibroblasts

Pluripotency relies on species-specific amino acid (AA) metabolism. In the mouse, inner cell mass (ICM) and ICM-derived pluripotent stem cells (PSCs) need threonine, which is catabolized by threonine dehydrogenase (TDH) into acetyl–CoA and glycine. Depleting (Δ) the culture medium of threonine (ΔT) or blocking TDH activity induces PSC death. By contrast, human PSCs do not survive without lysine (ΔK), leucine (ΔL), or methionine (ΔM). Since isolated bovine PSCs cannot be propagated in vitro, we screened for AAs that selectively support pluripotent ICM cells in intact bovine embryos. Five days (D5) post-IVF, embryos were transferred into glutamine-free synthetic oviduct fluid (gSOF) with Eagle’s nonessential (NE) and essential (E) AAs (gSOF_AA) plus BSA. Embryos were individually cultured until D8 under different conditions. Statistical significance was determined using Fisher’s exact test for blastocyst development (morphological grading to IETS standard) and t-tests for cell numbers (differential stain) and gene expression (quantitative or qPCR). Removal of BSA reduced grade 1–3 blastocyst (B1–3) development (37% v. 25%, n = 3; P < 0.001). Depleting NEAAs from gSOF_AA did not significantly decrease B1–3, but depleting all 12 EAAs did (25% v. 8%, n = 6; P < 0.001). Because ΔEAA was most effective, we focused on this. Experiments were conducted in gSOF+NEAA and compared with gSOF_AA as a positive control (n = 2–6 replicates). One (ΔT, ΔM), two (ΔMT, ΔCM, ΔCT; ΔIL, ΔIK, ΔKL), three (ΔCMT, ΔIKL), or six (ΔHPRVWY) EAA drop-out did not affect blastocyst formation, even when NEAAs were also removed for ΔT and ΔM groups (n = 3). However, depleting another six (ΔCIKLMT), nine (+CMT, +IKL), or eleven EAAs (+T, +M) increasingly compromised B1–3 (P < 0.05). Because no clear EAA candidates emerged from the screen, we focused on TDH. TDH mRNA was present at similar levels in microsurgically isolated (by microblade) trophectoderm (TE) and chemically isolated (by Triton X-100) ICM, but undetectable in five adult tissues. Despite ΔT medium showing no effect, exposure to the TDH inhibitor QC1 (50 µM) reduced B1–3 and B1–2 compared with a dimethylsulfoxide (DMSO) solvent control (25% v. 37% and 8% v. 19%, n = 8; P < 0.005). ICM and TE cell numbers were equally reduced in QC1 v. DMSO-treated blastocysts (10 v. 19 and 37 v. 67 with N = 21 and N = 29 embryos, respectively, n = 3; P < 0.005). Yet TDH, hypoblast (PDGRFα), epiblast (NANOG, FGF4, SOX2), and trophoblast (CDX2, KRT8) markers were not consistently affected by QC1. We next applied 3-hydroxynorvaline (3-HNV), which TDH hydrolyses into glycine and propionyl-CoA instead of acetyl-CoA. Compared with solvent controls, 3-HNV (300 µM) killed all embryos and bovine fetal fibroblasts within 3 days in ΔT medium. This toxic effect was fully rescued by >10-fold T-supplementation. Thus, 3-HNV protein incorporation, rather than acetyl-CoA reduction, may nonspecifically impair cellular function. In summary, we found that bovine ICM formation did not specifically depend on metabolizing threonine or any other single EAA. Research was supported by AgResearch Core Funding.

scholarly journals A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 142
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Apoptosis Rate ◽  
Cell Counts ◽  
Hatching Rates

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

scholarly journals Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1

2019 ◽  
Vol 97 (12) ◽  
pp. 4946-4950 ◽  
Author(s):  
Lydia K Wooldridge ◽  
Madison E Nardi ◽  
Alan D Ealy
Keyword(s):  
Embryo Culture ◽  
Zinc Supplementation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Retention Rates ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cell Numbers

Abstract Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P &lt; 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P &lt; 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

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.

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&lt;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&lt;0.005). In BSF, the re-expansion rate was 52% (28/54), which was significantly lower than in both BV and BC (both P&lt;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&gt;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&gt;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&gt;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.

scholarly journals Biological differences between in vitro produced bovine embryos and parthenotes

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Enrique Gómez ◽  
Alfonso Gutiérrez-Adán ◽  
Carmen Díez ◽  
Pablo Bermejo-Alvarez ◽  
Marta Muñoz ◽  
...  
Keyword(s):  
De Novo ◽  
Inner Cell Mass ◽  
Relative Proportion ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Biological Differences

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes andin vitroproduced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage.In vitromatured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-relatedPOU5F1and the methylationDNMT3Agenes were downregulated in parthenotes. Among pregnancy recognition genes,TP-1was upregulated in parthenotes, whilePGRMC1andPLAC8did not change. Expression ofp66shcandBAX/BCL2ratio were higher, andp53lower, in parthenotes. Among metabolism genes,SLC2A1was downregulated, whileAKR1B1,PTGS2,H6PD, andTXNwere upregulated in parthenotes, andSLC2A5did not differ. Among genes involved in compaction/blastulation,GJA1was downregulated in parthenotes, but no differences were detected withinATP1A1andCDH1. Within parthenotes, the expression levels ofSLC2A1,TP-1, andH6PD, and possiblyAKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, throughp66shcandp53respectively, and in their mechanisms to control pluripotency andde novomethylation.

156 IN VITRO DEVELOPMENT OF BOVINE MORULAE PRODUCED AND/OR CULTURED WITH ACTIVIN

2010 ◽  
Vol 22 (1) ◽  
pp. 236 ◽  
Author(s):  
B. Trigal ◽  
E. Gómez ◽  
C. Diez ◽  
J. N. Caamaño ◽  
I. Molina ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Total Cell ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Morula Stage ◽  
Vitro Development ◽  
And Control

We reported that the presence of activin during in vitro culture improves embryo development without changing the cell distribution in the blastocyst (Díez et al. 2009 AETE in press). In the present work, we aimed to analyze the morula stage as a putative milestone to activin exert differential effects. Day -5 morulae were produced with IVMFC oocytes from abattoir ovaries, using SOF with amino acids, myo-inositol, and 3 g L-1 of BSA as a culture medium. Embryo culture contained 10 ng mL-1 or 0 ng mL-1 of activin from Day -3 to Day -5. Early morulae (n = 543 out of 1099 cultured oocytes) were selected and subsequently cultured with or without 10 ng mL-1 of activin up to Day -8. Embryo development was daily monitored and cells differentially counted in Day -8 expanded blastocysts. (Thouas et al. 2001 Reprod. Biomed. 2001 3, 25-29). Data were analyzed by general linear model and presented as least squares means ± SEM. Activin from Days 3 to 5 did not change Day -5 morulae rates (P > 0.8). In morulae produced without activin (Days 5 to 8 and control), a treatment with activin from Days 5 to 8 improved total blastocyst rates v. controls, both in Day -7 and Day -8 (50.9 ± 3.6 v. 32.6 ± 3.6 and 60.8 ± 2.9 v. 42.3 ± 2.9, respectively; P < 0.01). Similarly, Day -7 expansion rates with activin (Days 5 to 8) were higher than controls (14.6 ± 1.8 v. 8.6 ± 1.8; P < 0.03). However, the above effects were not the same as those observed in morulae produced with activin (Days 3 to 5 and Days 3 to 8), where blastocyst development between activin treatment and controls only significantly differed in expansion rates on Day -7 (14.9 ± 1.8 v. 5.8 ± 1.8, respectively; P < 0.03). Morulae treated with activin (Days 5 to 8) yielded Day -7, total and expanded blastocyst rates, higher than morulae produced with activin (Days 3 to 5) (50.9 ± 3.6 v. 37.4 ± 3.6 and 14.6 ± 5.8 v. 5.8 ± 1.8, respectively; P < 0.03). Expansion rates on Day -8 were numerically higher within morulae produced and/or treated with activin (Days 3 to 8, Days 5 to 8, and Days 3 to 5) (values between 26.7 ± 2.6 and 27.4 ± 2.6) than in controls without activin at any time (19.2 ± 2.6) (P > 0.05). Trophectoderm (TE) cell numbers were reduced in embryos produced and/or treated with activin (Days 3 to 8, Days 3 to 5, and Days 5 to 8) (values between 109.4 ± 7.6 and 115.3 ± 7.9) as compared with untreated controls (141.2 ± 10.1) (P < 0.05). In morulae produced without activin, total cell counts were lower with activin being present from Day -5 to Day -8 (154.0 ± 8.8 v. 128.4 ± 7.2; P < 0.05). Inner cell mass (ICM) and ICM/total cell ratio were not affected by the presence of activin (P > 0.05). Activin did not change Day -5 morulae rates, although subsequent blastocyst development was in part affected by the presence of activin before the morula stage. Interestingly, improvements in blastocyst development, including expansion rates, triggered by activin led to reduced TE and unaltered ICM cell counts, suggesting that activin inhibits TE differentiation. Support: Cajastur (B. Trigal). MCINN: M. Muñoz (RYC08-03454); D. Martín (PTA2007-0268-I); INIA (I. Molina); Project HF2007-0126.

218 DEVELOPMENT OF EVENLY AND UNEVENLY CLEAVED TWO-CELL PORCINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 188
Author(s):  
D. N. Q. Thanh ◽  
K. Kikuchi ◽  
T. Somfai ◽  
M. Ozawa ◽  
M. Nakai ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Great Influence ◽  
Total Cell ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Further Development

Mammalian eggs are so microlecithal that the embryos would be expected to divide in unison and that each division would lead to 2 equal blastomeres, which are believed to have a greater competence for further development than zygotes with unequal cleavage. However, some studies have shown that uneven blastomere size commonly occurs from the very first division in mammals, and it seems to be concerned with the generation of the first cell lineages of the blastocyst cells: trophectoderm and the inner cell mass (Gueth-Hallonet and Maro 1992 Trends Genet. 8, 274–279). In our study, we produced porcine embryos in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1031–1041), and newly formed 2-cell embryos were collected. Based on the timing of the first cleavage (30 or 36 h after insemination), the cleavage pattern (E: equal; U: unequal) and the presence or absence of a second cleavage (+ or –) within the first 2 days of IVC was classified into groups: 30E(–), 30E(+), 30U(–), 30U(+), 36E(–), 36E(+), 36U(–), or 36U(+). There was no difference between the 30E and 30U groups in proportions of the 2-cell stage, which had a nucleus in both blastomeres (99.0 � 0.8% and 91.4 � 3.6%, respectively) or between the 36E and 36U groups (98.2 � 1.1% and 88.0 � 7.2%, respectively). Comparison of further development between the 30E and 30U groups showed that there was no difference in blastocyst rates (70.7 � 5.7% and 61.7 � 7.8%, respectively) and total cell numbers (39.1 � 2.1 and 31.7 � 2.3, respectively). Although the blastocyst rate in the 36E group (37.3 � 6.7%) was significantly higher (P < 0.05) than that of the 36U group (12.0 � 5.1%), the total cell number was not different (26.3 � 5.5 and 25.3 � 5.2, respectively). The timing of the first division, however, had a great influence on further development of the embryos; the 30-h cleaved embryos had a greater rate of blastocyst development (68.2 � 6.3%) than did the 36-h embryos (28.2 � 4.8%, P < 0.01 by ANOVA). The cell numbers of blastocysts derived from 30-h cleaved embryos (37.2 � 2.6) were significantly higher than those of the 36-h embryos (26.2 � 2.3, P < 0.01) as well. Two-cell embryos that were newly formed at 30 h and underwent the next cleavage within the first 2 days of IVC (30 + group) had a higher blastocyst rate (74.8 � 7.0%) and greater cell numbers (40.6 � 2.6) than those not showing a second division during this period (30– group; 46.8 � 5.0% and 19.9 � 2.2, respectively). In contrast, for embryos showing the first cleavage at 36 h of insemination, the presence of the next cleavage within 2 days after the first cleavage did not have any effect on embryonic development. These results suggest that the developmental ability of porcine embryos was influenced by the timing and shape of the first cleavage and by the subsequent occurrence of the second cleavage.

scholarly journals 127 DEVELOPMENT AND QUALITY OF BOVINE MORULAE CULTURED IN SERUM-FREE MEDIUM WITH RETINOIC RECEPTOR SPECIFIC AGONISTS

2008 ◽  
Vol 20 (1) ◽  
pp. 144
Author(s):  
E. Gómez ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
A. Rodríguez ◽  
N. Facal ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Inner Cell Mass ◽  
Retinoic Acid Receptor ◽  
Cell Mass ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Counts ◽  
All Trans Retinoic Acid ◽  
Apoptosis And Necrosis

In the cell, all-trans retinoic acid (ATRA), a vitamin A metabolite, binds to retinoic acid-receptor (RAR), whereas the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). Synthetic compounds such as LG100268 (LG; Ligand Laboratories) are highly specific to bind RXR, which allows to differentially study the RAR and RXR pathways. In previous work morulae treated with LG for 48 h showed to improve blastocyst development and to activate pro-apoptotic genes (in press), whereas ATRA for 24 h increased cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodr�guez et al. 2006 Hum. Reprod. 21, 2149–2157). However, LG and ATRA were never both compared for 24 in medium with BSA, which is thought to be more appropriate to produce embryos for cryopreservation than serum-containing medium. In this work we analyze development, quality, and viability of morulae cultured with RAR and RXR agonists. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in synthetic oviduct fluid (SOF) +3 gL–1 BSA. On day 6, morulae were treated for 24 h with ATRA 0.7 µm, LG 0.1 µm, or no additives. Blastocyst development was monitored up to day 8. Differential cell counts were made on hatched blastocysts on days 7 and 8. Apoptosis and necrosis (TUNEL + nuclear histology) were made on day 8 expanded and hatched blastocysts. Data were analyzed by GLM and Duncan's test, expressed as LSM � SE, and development rates were expressed as percentages of cultured morulae (replicates [R] = 14 for development; R = 9 for cell counts; R = 4 for apoptosis; n = 1647 morulae). ATRA yielded more blastocysts on day 8 than LG and controls (72.2 � 2.2 v. 60.0 � 2.3 and 65.6 � 2.4, respectively; P < 0.02), and more expanded blastocysts than LG (48.6 � 2.3 v. 36.6 � 2.4; P < 0.02), but no more than controls (43.5 � 2.5). Day-7 and day-8 hatched blastocysts cultured with ATRA showed more total cells than day-7 controls (163.5 � 8.0 and 161.5 � 5.4 v. 137.7 � 8.9, respectively; P < 0.05). However, in the presence of ATRA, day-8 blastocysts showed a strong cell reduction in the inner cell mass (ICM), whereas their day-7 counterparts conserved ICM/total cells proportions comparable to day-7 controls (11.0 � 1.2 v. 19.7 � 1.7 and 20.6 � 1.9, respectively; P < 0.03). The LG increased apoptotic index (AI) and necrotic index (NI) in the ICM (AI: 14.5 � 2.4 v. 6.4 � 1.5 and 6.4 � 1.4; NI: 5.0 � 1.2 v. 0.9 � 0.8 and 1.6 � 0.7; for LG, ATRA, and controls, respectively; P < 0.02). Embryos produced with ATRA showed improved development and cell distribution without increasing apoptosis and necrosis. Vitrification of excellent day-7 and day-8 blastocysts is in course to evaluate cryosurvival and further embryo transfer to determine full developmental competence. Grant Support: MEC, project AGL2005-04479. M. Muñoz is sponsored by FICYT.

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