scholarly journals Lysophosphatidic Acid Accelerates Bovine In Vitro-Produced Blastocyst Formation through the Hippo/YAP Pathway

2021 ◽  
Vol 22 (11) ◽  
pp. 5915
Author(s):  
Bo Yu ◽  
Helena T. A. van Tol ◽  
Christine H. Y. Oei ◽  
Tom A. E. Stout ◽  
Bernard A. J. Roelen
Keyword(s):  
Signaling Pathway ◽  
Lysophosphatidic Acid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Hippo Signaling ◽  
Blastocyst Formation ◽  
Cdx2 Expression ◽  
Gene And Protein Expression

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.

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.

99 EFFECT OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON IN VITRO-PRODUCED PORCINE OOCYTES AND THEIR SUBSEQUENT DEVELOPMENT: A PARTHENOGENETIC STUDY

2008 ◽  
Vol 20 (1) ◽  
pp. 130
Author(s):  
D. Biswas ◽  
J. H. Lee ◽  
E. B. Jeung ◽  
E. S. Lee ◽  
S. H. Hyun
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Inner Cell Mass ◽  
Formation Rate ◽  
Cell Mass ◽  
Vascular Endothelial ◽  
Blastocyst Formation ◽  
Group B ◽  
Endothelial Growth Factor

The addition of vascular endothelial growth factor (VEGF) to maturation media has beneficial effects on oocyte maturation and blastocyst formation (Einspanier et al. 2002 Mol. Reprod. Dev. 62, 29–36). The present study was conducted to examine the effect of parthenogenesis on in vitro-matured porcine oocytes with VEGF along with porcine follicular fluid in the maturation media. Porcine ovaries were collected from a local slaughter house in physiological saline. After aspiration, COC were matured in vitro in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and (1) Group A: 10% pFF; (2) Group B: 10% pFF and 5 ng mL–1 of VEGF; (3) Group C: 10% polyvinyl alcohol; or (4) Group D: 5 ng mL–1 of VEGF plus 10% polyvinyl alcohol. Fifty COC were cultured for the first 22 h at 390�C in a humidified atmosphere of 5% CO2 in 95% air with 4 IU mL–1 of eCG and 4 IU mL–1 of hCG. They were then transferred to hormone-free medium and cultured for an additional 20 h. After culture, COC were denuded with hyaluronidase, and a proportion were stained with Hoechst 33342 for evaluating the metaphase II stage. The remaining oocytes were subjected to electrical parthenogenesis by using a 1-mm fusion chamber and were activated by applying 2 direct current pulses of 110V for 60 µs. Cleavage and blastocyst formation rate were evaluated under a stereomicroscope at 48 and 168 h after activation, respectively. Blastocyst quality was assessed by differential staining of inner cell mass and trophectoderm cells according to a modified staining procedure (Thouas et al. 2001 Reprod. Biomed. Online 3, 25–29). All data are presented as mean � SD and were analyzed by ANOVA followed by Duncan's multiple range test using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The maturation rate was significantly higher (P < 0.05) in Groups A and B than Groups C and D (76.1 � 9.6, 78.9 � 6.0 v. 60. � 14.2 and 58.3 � 14.3, respectively). The cleavage rate was significantly higher (P < 0.05) in Groups A and B (73.2 � 1.8 and 64.6 � 1.1, respectively) than Groups C and D (47.9 � 1.8 and 48.3 � 1.7, respectively). The blastocyst formation rate was significantly higher (P < 0.05) in Group B (32.6 � 2.4) compared to other groups. There was no significant difference in blastocyst cell number (inner cell mass or trophectoderm) among these groups. These data indicate that the exogenous VEGF along with pFF in the maturation media helps to increase the blastocyst formation rate in vitro, and it might be due to presence of some ligand/protein kinase in the pFF that plays an important role during the cyclic growth of oocytes.

scholarly journals Increased apoptosis in bovine blastocysts exposed to high levels of IGF1 is not associated with downregulation of the IGF1 receptor

Reproduction ◽  
2011 ◽  
Vol 141 (1) ◽  
pp. 91-103 ◽  
Author(s):  
M A Velazquez ◽  
D Hermann ◽  
W A Kues ◽  
H Niemann
Keyword(s):  
Protein Expression ◽  
Inner Cell Mass ◽  
Polycystic Ovary ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Preimplantation Mouse ◽  
High Concentrations ◽  
Igf1 Receptor

The hypothesis that high concentrations of IGF1 can impair embryo development was investigated in a bovine in vitro model to reflect conditions in polycystic ovary syndrome (PCOS) patients. Embryos were either cultured in the absence or presence of a physiological (100 ng/ml) or supraphysiological (1000 ng/ml) IGF1 concentration. Cell allocation, apoptosis, transcript and protein expression of selected genes involved in apoptosis, glucose metabolism and the IGF system were analysed. Supraphysiological IGF1 concentration did not improve blastocyst formation over controls, but induced higher levels of apoptosis, decreased TP53 protein expression in the trophectoderm and increased the number of cells in the inner cell mass (ICM). The increase in ICM cells corresponded with an increase in IGF1 receptor (IGF1R) protein in the ICM. A small, but significant, percentage of blastocysts displayed a hypertrophic ICM, not observed in controls and virtually absent in embryos treated with physiological concentrations of IGF1. Physiological IGF1 concentrations increased total IGF1R protein expression and upregulated IGFBP3 transcripts leading to an increase in blastocyst formation with no effects on cell number or apoptosis. In conclusion, the results support the hypothesis of detrimental effects of supraphysiological IGF1 concentrations on early pregnancy. However, our results do not support the premise that increased apoptosis associated with high levels of IGF1 is mediated via downregulation of the IGF1R as previously found in preimplantation mouse embryos. This in vitro system with the bovine preimplantation embryo reflects critical features of fertility in PCOS patients and could thus serve as a useful model for in-depth mechanistic studies.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

scholarly journals The Hippo Signaling Pathway Components Lats and Yap Pattern Tead4 Activity to Distinguish Mouse Trophectoderm from Inner Cell Mass

2009 ◽  
Vol 16 (3) ◽  
pp. 398-410 ◽  
Author(s):  
Noriyuki Nishioka ◽  
Ken-ichi Inoue ◽  
Kenjiro Adachi ◽  
Hiroshi Kiyonari ◽  
Mitsunori Ota ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Hippo Signaling ◽  
Hippo Signaling Pathway

scholarly journals 50SPATIAL EXPRESSION OF OCT4 IS NORMALLY REGULATED IN PREIMPLANTATION STAGE BOVINE SOMATIC CELL CLONES

2004 ◽  
Vol 16 (2) ◽  
pp. 147
Author(s):  
S. Kurosaka ◽  
S. Eckardt ◽  
M.K. Friez ◽  
N.A. Leu ◽  
R. Reinbold ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Inner Cell Mass ◽  
Marker Gene ◽  
Cell Mass ◽  
Calf Serum ◽  
Ectopic Expression ◽  
Cell Lineage ◽  
Embryonic Cells ◽  
Oct4 Protein

Oct4 is a member of the POU family of transcription factors and is expressed in blastomeres, pluripotent embryonic cells and the germ cell lineage. In the mouse blastocyst, expression of Oct4 becomes restricted to the inner cell mass (ICM). In bovine, Oct4 has not been considered to be a marker for pluripotency because Oct4 protein is expressed in both the ICM and the trophectoderm (TE) of bovine blastocysts (van Eijk MJT et al., 1999 Biol. Reprod. 60, 1093–1103; Kirchhof N et al., 2000 Biol. Reprod. 63, 1698–1705). Oct4 has been used as a marker gene for nuclear reprogramming in cloned embryos. Aberrant spatial distribution and levels of Oct4 have been observed in the majority of mouse clone blastocysts and blastocyst outgrowths (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), indicating reprogramming failure of mouse clones. Lack of or abnormal Oct4 protein expression was also observed in cloned monkey embryos (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159). The spatial distribution of Oct4 mRNA and protein in bovine clones has not been reported. Bovine oocytes were obtained from a commercial supplier (BOMED, Inc., Madison, WI, USA), and were matured in vitro. Enucleated oocytes were fused with fibroblasts from ear skin and then treated with 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D for 6h. Embryos were cultured in SOF supplemented with 1% fetal calf serum (FCS) at 39°C under 5% CO2, 5% O2 and 90% N2 for 2 days. At Day 2, embryos were transferred to SOF supplemented with 5% FCS and cultured under the same conditions until Day 7. Blastocysts were analyzed at Day 7. Oct4 mRNA expression was visualized by whole-mount in situ hybridization using a bovine Oct4-specific antisense riboprobe. Oct4 protein was detected by immunocytochemistry. Control embryos were produced by IVF and were cultured under the same conditions to the blastocyst stage (Day 7). We found that Oct4 mRNA signal was restricted to the ICM in bovine blastocysts. Bovine clones were not different from control embryos in that distribution of Oct4 mRNA signal was typically restricted to the ICM (14 of 16). In contrast to our previous report on mouse clones (Boiani M et al., 2002 Genes Dev. 16, 1209–1219), ectopic expression of Oct4 mRNA in the TE was rarely detected in bovine clones (2 of 16). Distribution of Oct4 protein was also similar between clones and controls with distribution in both the ICM and TE (clones: 9 of 9; controls: 9 of 9). It is unclear why defects in Oct4 distribution should differ between bovine and other species tested including monkey (Mitalipov SM et al., 2003 Biol. Reprod. 68 (suppl 1), 159–160); however, the higher rate of normal Oct4 distribution is consistent with the generally higher rates of postimplantation development of bovine clones (Shi W et al., 2003 Differentiation 71, 91–113).

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