scholarly journals ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1914 ◽  
Author(s):  
Jeongwoo Kwon ◽  
Nam-Hyung Kim ◽  
Inchul Choi
Keyword(s):  
Tight Junction ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Coiled Coil ◽  
Specific Inhibitor ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Genes Encoding ◽  
Rock Activity

The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P< 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins.

scholarly journals PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1272 ◽  
Author(s):  
Muhammad Idrees ◽  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Mrna Translation ◽  
Bovine Oocyte ◽  
Blastocyst Development

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

scholarly journals Modified Spirulina maxima Pectin Nanoparticles Improve the Developmental Competence of In Vitro Matured Porcine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2483
Author(s):  
Pantu-Kumar Roy ◽  
Ahmad-Yar Qamar ◽  
Bereket-Molla Tanga ◽  
Seonggyu Bang ◽  
Gyeonghwan Seong ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Spirulina Maxima ◽  
Blastocyst Development ◽  
Parthenogenetic Activation ◽  
Molecular Approaches

Molecular approaches have been used to determine metabolic substrates involved in the early embryonic processes to provide adequate culture conditions. To investigate the effect of modified Spirulina maxima pectin nanoparticles (MSmPNPs) on oocyte developmental competence, cumulus–oocyte complexes (COCs) retrieved from pig slaughterhouse ovaries were subjected to various concentrations of MSmPNPs (0, 2.5, 5.0, and 10 µg/mL) during in vitro maturation (IVM). In comparison to the control, MSmPNPs-5.0, and MSmPNPs-10 groups, oocytes treated with 2.5 µg/mL MSmPNPs had significantly increased glutathione (GSH) levels and lower levels of reactive oxygen species (ROS). Following parthenogenetic activation, the MSmPNPs-2.5 group had a considerably higher maturation and cleavage rates, blastocyst development, total cell number, and ratio of inner cell mass/trophectoderm (ICM:TE) cells, when compared with those in the control and all other treated groups. Furthermore, similar findings were reported for the developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos. Additionally, the relative quantification of POU5F1, DPPA2, and NDP52 mRNA transcript levels were significantly higher in the MSmPNPs-2.5 group than in the control and other treated groups. Taken together, the current findings suggest that MSmPNP treatment alleviates oxidative stress and enhances the developmental competence of porcine in vitro matured oocytes after parthenogenetic activation and SCNT.

scholarly journals Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 345-355 ◽  
Author(s):  
F Moulavi ◽  
S M Hosseini ◽  
M Hajian ◽  
M Forouzanfar ◽  
P Abedi ◽  
...  
Keyword(s):  
Cell Fate ◽  
Embryo Development ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Nuclear Remodeling

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep.In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances ofHSP90AA1(HSP90),NPM2andATPasegenes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents ofPOU5F1(OCT4) with the ZI-NT and ZF-NT methods and ofPAPOLA(PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

scholarly journals Melatonin Alleviates the Toxicity of High Nicotinamide Concentrations in Oocytes: Potential Interaction with Nicotinamide Methylation Signaling

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Marwa El-Sheikh ◽  
Ahmed Atef Mesalam ◽  
Seok-Hwan Song ◽  
Jonghyeok Ko ◽  
Il-Keun Kong
Keyword(s):  
Inner Cell Mass ◽  
Active Form ◽  
Cell Mass ◽  
Potential Interaction ◽  
Developmental Competence ◽  
Ros Scavenging ◽  
Vitamin B3 ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10−7 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.

82 HOW LOW CAN YOU GO? DEFINING THE MINIMAL NUTRIENT REQUIREMENTS FOR BOVINE EMBRYOS IN CULTURE

2017 ◽  
Vol 29 (1) ◽  
pp. 148
Author(s):  
J. R. Herrick ◽  
A. F. Greene ◽  
J. Becker ◽  
W. B. Schoolcraft ◽  
R. L. Krisher
Keyword(s):  
Fatty Acid ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Nutrient Concentrations ◽  
Acid Oxidation ◽  
Control Medium ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Blastocyst Development

Recent metabolomic studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients provided to them in culture media. Our objective was to determine the effects of reducing nutrient concentrations in our media to 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of those in our control medium (100%) on the development of in vitro-matured/IVF bovine embryos in a serum-free medium. Cumulus–oocyte complexes were matured in a defined maturation medium (0.1 IU mL−1 recombinant human FSH, 50 ng mL−1 recombinant mouse epidermal growth factor, and 2.5 mg mL−1 recombinant human serum albumin) and co-incubated with frozen–thawed spermatozoa (2 × 106 mL−1, 20 h). Embryos were cultured (7.5% CO2, 6.5% O2, 38.7°C) in a sequential media system (0–72 h and 72–168 h). Concentrations of salts, bicarbonate, and protein (2.5 mg mL−1 fatty acid–free BSA) were the same in all treatments. All nutrients (glucose, lactate, pyruvate, amino acids, and vitamins) were diluted to the same extent (e.g. 25%) relative to the control medium for each culture period. Blastocyst formation and hatching (per cleaved embryo) were evaluated on Day 7 of culture. Hatching blastocysts were stained to determine the number of inner cell mass (ICM; SOX2+), trophectoderm (TE; CDX2+), and total cells (ICM+TE) in the embryo. All data were analysed by ANOVA. The proportion of zygotes that cleaved on Day 3 was not affected (P > 0.05) by the concentration of nutrients present. In experiment 1, dilution of nutrients to 25% did not affect (P > 0.05) blastocyst development (40.1 ± 3.7%) or hatching (16.3 ± 2.8%) compared with 100% (45.2 ± 3.8% blastocyst and 24.9 ± 3.3% hatching). In experiment 2, dilution of nutrients to 12.5% tended (P = 0.08) to reduce hatching (12.9 ± 2.6%) compared with 100% (20.0 ± 3.1%) but did not affect (P > 0.05) blastocysts formation (12.5%, 41.7 ± 3.9% v. 100%, 40.0 ± 3.8%). It was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05). Hatching blastocysts cultured with 25 or 12.5% nutrients had fewer total (P < 0.05; 150.7 ± 9.7 and 121.6 ± 7.6, respectively), TE (P < 0.05; 124.1 ± 8.5 and 90.5 ± 7.1), and ICM (P = 0.06; 26.6 ± 3.4 and 30.7 ± 4.0) cells compared with control embryos (195.2 ± 15.9 total, 156.1 ± 14.1 TE, and 39.1 ± 4.0 ICM). To determine whether the embryo’s ability to develop with reduced concentrations of nutrients was dependent on lipid metabolism, embryos were cultured with 50, 25, 12.5, and 6.25% nutrients in the presence or absence of an inhibitor of fatty acid oxidation (50 μM etomoxir). The presence of etomoxir reduced (P < 0.05) blastocyst development at all nutrient concentrations, but this effect was more pronounced when nutrients were limited (≤25% nutrients, 28.7 to 40.9% reduction) compared with 50% (12.5% reduction). Although blastocyst cell numbers decrease when nutrient concentrations are reduced to 25% of those in control media, the proportion of embryos reaching the blastocyst stage is not affected until nutrients are reduced to 6.25%. The ability to develop under nutrient-restricted conditions appears to be related to fatty acid metabolism.

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.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo

Reproduction ◽  
2021 ◽  
Vol 161 (4) ◽  
pp. 353-363
Author(s):  
Mun-Hyeong Lee ◽  
Pil-Soo Jeong ◽  
Bo-Woong Sim ◽  
Hyo-Gu Kang ◽  
Min Ju Kim ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Early Phase ◽  
Inner Cell Mass ◽  
Formation Rate ◽  
Cell Mass ◽  
Female Reproductive Tract ◽  
Developmental Competence ◽  
Early Embryos ◽  
Developmental Parameters

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0–2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2–4 days) and late phases (4–6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

scholarly journals 173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 237 ◽  
Author(s):  
G. Lazzari ◽  
I. Lagutina ◽  
G. Crotti ◽  
P. Turini ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Es Cell ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
In Vivo Culture

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

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