147 AGGREGATION OF IN VIVO FERTILIZED AND PARTHENOGENETICALLY ACTIVATED MOUSE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 221
Author(s):  
H. Fernandes ◽  
P. T. Mihara ◽  
B. Cazari ◽  
I. P. Emanuelli ◽  
M. F. G. Nogueira
Keyword(s):  
Cell Mass ◽  
Blastocyst Stage ◽  
Strontium Chloride ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Fisher’S Exact Test ◽  
Inverted Microscope ◽  
Parthenogenetic Embryos

Embryonic chimerism – mixing of cells originated from at least two different fertilizations – has been used as a tool for stem cell pluripotency diagnosis, transgenic rodent production, and organogenesis studies. Additionally, parthenotes are used for studies related to gene imprinting and the ability of their cells to compose the placenta and/or an adult animal. The aim of this work was to validate the production and characterize developmental kinetic of parthenogenetic embryos, obtained from C57BL/6 EGFP mice (EGFP), to determine their potential to produce chimeras, and to localize parthenogenetic cells on the produced blastocyst. Embryos were harvested from superovulated females. For the aggregation, pre-compaction IVF (Swiss Webster/SW strain) or parthenogenetic activated (PGA; SW and EGFP) embryos were used. For PGA, strontium chloride hydrate (5 mM for 6 h) was used. Two experiments were outlined in order to: i) evaluate the development and kinetic from IVF (IVF and in vitro developed; 2-cell stage embryos, n = 53) and from PGA (parthenogenetically activated and in vitro developed; n = 409 oocytes) techniques, both from SW; ii) evaluate the aggregation between pairs of control embryos (C; n = 20, 4-cell in vivo produced embryos from SW) or pairs of 4-cell in vivo produced SW embryos and 4- to 8-cell PGA EGFP embryos (parthenogenetic embryo, PG; n = 40). After manipulation (removal of the zona pellucida and approximation of pairs, for C and PG groups), all the groups were kept in vitro culture (37°C, 5% CO2, and saturated humidity) for 48 to 60 h (C and PG), or up to blastocyst stage (IVF and PGA). Chimerism rate and parthenogenetic cells fluorescence (pre- and post-culture) were evaluated under an inverted microscope with epifluorescence source, and digital images were captured (Eclipse Ti and NIS-Elements, Nikon, Japan, respectively), by merging visible and UV light images. The rate of PGA (EGFP oocytes) was 54.5% (66/121), assessed 6 h post-activation by the presence of at least one pronucleus. The assessment was performed with 20× magnification and Hoffman modulation contrast in an inverted microscope. The rate of blastocyst production between IVF and PGA was significantly different (71.4 and 12.9%, respectively; P < 0.001, Fisher’s exact test). When developmental kinetic was evaluated there a difference in the average time to the majority of embryos reach blastocyst stage for IVF (48 h) and PGA (120 h) was observed. The chimerism rate was assessed by the presence of a single and cohesive cell mass (C) or by the incorporation of EGFP cells in the SW embryo (PG). There was difference (P = 0.006; Fisher’s exact test) between C (55.0%; 11/20) and PG (17.5%; 7/40) chimerism rates, assessed from 48 to 60 h of culture. In PG group, the incorporation of EGFP cells in the obtained chimeras was mainly detected in the trophectoderm although one chimeric blastocyst contained EGFP cells only in the inner cell mass. It was concluded that parthenogenetic embryos have a slower developmental kinetic (until 120 h of culture) than embryos derived from IVF. Acknowledgment to FAPESP for fellowship and funding.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

Δράση του φυτού βαλσαμόχορτο (Hypericum perforatum) στον καρκίνο της ουροδόχου κύστης

2019 ◽  
Author(s):  
Ιωάννης Τσίμαρης
Keyword(s):  
Hypericum Perforatum ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Rp Hplc ◽  
Fisher’S Exact Test

Σκοπός της παρούσας διδακτορικής διατριβής ήταν η συστηματική και εις βάθος έρευνα της αντικαρκινικής δράσης του φυτού βαλσαμόχορτου γνωστού και ως Hypericim Perforatum (HP) κατά του καρκίνου της κύστης. Με την βοήθεια υγρής χρωματογραφίας υψηλής απόδοσης αντίστροφης φάσης (RP-HPLC) αναγνωρίστηκε ότι το κύριο συστατικό του υδατικού διαλύματος που λαμβάνεται στα πλαίσια της παραδοσιακής ηπειρώτικης ιατρικής είναι η υπερφορίνη (2,36 ± 0,03 mg/g).Τοπειραματικό μοντέλο περιλάμβανε αρσενικά ποντίκια BULBc τα οποία εκτέθηκαν σε νιτροζαμίνη (OH-BBN) σε συγκέντρωση 1000ppm μέρα παρά μέρα για 6 εβδομάδες. Θυσιάστηκαν μετά από 26 εβδομάδες. Τα ιστολογικά δεδομένα έδειξαν ότι το υδατικό διάλυμα ασκεί στατιστικώς σημαντική αντικαρκινική δράση (Fisher’s exact test p=0.048) όταν χορηγείται για 26 εβδομάδες αφού επιδράσει ο καρκινογόνος παράγοντας. Η χορήγηση του εκχυλίσματος το διάστημα που λάμβαναν νιτροζαμίνη δεν εμφάνισε σημαντική αντικαρκινική δράση (Fisher’s exact test p=0.093). Στην συνέχεια προχωρήσαμε στην αναζήτηση του πιο δραστικού παραγώγου του φυτού, είτε με μορφή διαλυμάτων, είτε με την μορφή εκχυλισμάτων, είτε μετά από κλασματοποίηση. Η ανάλυση των συστατικών τους έγινε με RP-HPLC. Η αντικαρκινική δράση αποτιμήθηκε σε καρκινικές κυτταρικέςσειρές ουροδόχου κύστεως Τ24 και NBT-II σε συνθήκες σκότους. Η διαδικασία κλασματοποίησης ανέδειξε το έκτο κατά σειρά κλασματοποίησης κλάσμα του Εκχυλίσματος Πετρελαϊκού Αιθέρα (ΕΠΑ6) ως το πλέον δραστικό (LC50 0.17 μg/mL). Στην αναζήτηση των συστατικών που ευθύνονται για την αντικαρκινική δράση διαπιστώθηκε ότι αυτή δεν οφείλεται στην υπερφορίνη αλλά σε άγνωστες ουσίες οι οποίες δεν ήταν δυνατόν να ταυτοποιηθούν στην δεδομένη χρονική στιγμή. Ηδοκιμασία TUNEL απέδειξε πως ο κυτταρικός θάνατος επέρχεται μέσω της απόπτωσης. Από την φυσικοχημική ανάλυση των εκχυλισμάτων διαπιστώθηκε πως το Πολικό Μεθανολικό Κλάσμα (ΠΜΚ), σε μορφή πούδρας, περιέχει υπερικίνη σε συγκέντρωση 0.57% μόνο, είναι υδατοδιαλυτό, σταθερό σε θερμοκρασία δωματίου καθιστώντας το ενδιαφέρον φωτοευαισθητοποιητή για περαιτέρω μελέτη.Προχωρήσαμε σε σύγκριση του ΠΜΚ, σε διάφορες συγκεντρώσεις με το Photofrin® , εγκεκριμένη από το FDA ουσία για την Φωτοδυναμική Θεραπεία (ΦΔΘ). Το ΠΜΚ στη συγκέντρωση των 60 μg/mL, προκαλούσε κυτταρικό θάνατο στο 86% και στο 80% των Τ24 και RT4 καρκινικών κυττάρων αντίστοιχα. Το Photofrin® εμφάνισε κυτταρικό θάνατο στο 77% των Τ24 μόνο. Μετά από σύγκριση πηγών φωτισμού λευκού φωτός και ΠΜΚ σε Τ24 κύτταρα διαπιστώθηκε ότι η πιο αποτελεσματική ήταν η πηγή ψυχρού φωτισμού με λάμπα αλογόνου (Storz 482 Cold Light Fountain). Πειραματιζόμενοι με θηλυκούς Wistar αρουραίους με ποικίλες ενδοκυστικές δόσεις ΠΜΚ, με κλιμακούμενους χρόνους ενδοκυστικής παραμονής και ποικίλες ενέργειες διέγερσης καταλήξαμε στην δοσολογία των 36.5mg/ml του ΠΜΚ, δεκαπεντάλεπτη ενδοκυστική παραμόνή και διέγερση με 700J/cm2 λευκού φωτός. Με βάση το παραπάνω προχωρήσαμε σε πειραματικό μοντέλο ορθότοπης εμφύτευσης ουροθηλιακών καρκινικών κυττάρων ΑΥ27. Είχαμε πλήρη (100%) εξάλειψη επιφανειακών καρκινικών βλαβών αλλά και του φυσιολογικού ουροθηλίου στις 30 ημέρες μετά την θεραπεία. Το ουροθήλιο αναγεννάτε πλήρως, χωρίς την ύπαρξη ουλών, 90 ημέρες μετά την φωτοδυναμική θεραπεία. Συμπερασματικά το εκχύλισμα του υπέρικου, στη δοσολογία που το λάμβαναν οι ασθενείς, παρουσιάζει στατιστικώς σημαντική αντικαρκινική δράση όταν χορηγείταιμετά την δράση του καρκινογόνου. Η αντικαρκινική δράση που παρατηρείται in vitro στο αποδίδεται σε άγνωστες, προς το παρόν ουσίες, που περιέχονται στο ΕΠΑ6. Το ΠΜΚ εμφανίζει σημαντική αντικαρκινική δράση τόσο in vitro, μετά από διέγερση με λέιζερ ή λευκό φως, όσο και in vivo, με πλήρη εξάλειψη των καρκινικών λαβών, χωρίς την εμφάνιση ουλών στην ουροδόχου κύστη με πλήρως αναγεννημένο του ουροθήλιο μετά από 90 ημέρες.

178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 168
Author(s):  
L. Magnani ◽  
R. Cabot
Keyword(s):  
Gene Expression ◽  
Similar Pattern ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Developmental Expression ◽  
Fertilization In Vitro ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Parthenogenetic Embryos

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 43-55
Author(s):  
J. Rossant ◽  
K. M. Vijh
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Parietal Endoderm ◽  
Trophoblast Giant Cells ◽  
Vitro Development ◽  
Ectoplacental Cone

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6·5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8·5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5·5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

Abstract 4: Porcine Parthenotes as a Model to Evaluate Developmental Potential of Human Inducible Pluripotent Stem Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Naoko Koyano-Nakagawa ◽  
James Dutton ◽  
Mary G Garry ◽  
Daniel J Garry
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Parthenogenetic Embryos

The use of human induced pluripotent stem cells (hiPSCs) has tremendous potential for regenerative medicine by providing an unlimited source of personalized cells. A number of protocols have been established for efficient differentiation of hiPSCs to the desired lineage in vitro, such as cardiomyocytes and blood. However, the field lacks an in vivo system to evaluate the differentiation potential and quality of hiPSCs. Developmental potential of stem cells derived from experimental animals can be readily assessed by generating blastocyst chimeras and examination of the contribution to the embryos, or by the potential of teratoma formation. However, this is not possible in the case of humans. As a potential solution for this issue, we examined whether porcine parthenotes could be used as an experimental model to test the developmental potential of the hiPSCs. Parthenotes are generated by electrical activation of the oocytes collected at the abattoir and will develop up to gestational day 53 if transferred to a pseudo-pregnant sow. The embryonic culture conditions have also been established and the zygotes can develop normally to the expanded blastocyst stage (day 7 post fertilization/activation), in vitro. We took advantage of this in vitro system and examined the ability of hiPSCs to proliferate and integrate into the parthenogenetic embryos. Parthenogenetic embryos were injected with ten undifferentiated hiPSCs at day 4 (8 cell ~ morula stage) and cultured up to 72 hours. During this period, parthenotes underwent blastocoel cavity formation and hatching. Cell tracing experiments demonstrated that hiPSCs proliferated and integrated into the parthenotes. They retained pluripotency marker expression during this period. hiPSCs and their derivatives were found both in trophoectoderm and embryo proper. We further observed that the hiPSCs underwent cellular proliferation and promoted developmental progression of the parthenote in vitro. In summary, the porcine parthenote model system is an efficient high throughput system to examine the developmental capacity of human stem cell populations.

scholarly journals Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos

Reproduction ◽  
2002 ◽  
pp. 507-515 ◽  
Author(s):  
TJ King ◽  
J Zhu ◽  
HA Finlayson ◽  
W Bosma ◽  
L Harkness ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Model System ◽  
Developmental Potential ◽  
Maintenance Strategies ◽  
First Time ◽  
Parthenogenetic Embryos

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

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