scholarly journals ZP4 confers structural properties to the zona pellucida essential for embryo development

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ismael Lamas-Toranzo ◽  
Noelia Fonseca Balvís ◽  
Ana Querejeta-Fernández ◽  
María José Izquierdo-Rico ◽  
Leopoldo González-Brusi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Structural Properties ◽  
Zona Pellucida ◽  
Evolutionary Conservation ◽  
Protective Capacity ◽  
In Vitro Development ◽  
Fourth Component ◽  
Vitro Development

Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

scholarly journals Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
In Vitro Development ◽  
Porcine Islets ◽  
Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

scholarly journals Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+Lymphocytes with a Th2 Cytokine Profile

2000 ◽  
Vol 164 (6) ◽  
pp. 3047-3055 ◽  
Author(s):  
Dragana Jankovic ◽  
Marika C. Kullberg ◽  
Nancy Noben-Trauth ◽  
Patricia Caspar ◽  
William E. Paul ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Cytokine Profile ◽  
Cell Analysis ◽  
In Vitro Development ◽  
Th2 Cytokine ◽  
Vitro Development ◽  
Cd4 Lymphocytes

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

scholarly journals Bioreactors as physiologicallike in vitro models

2020 ◽  
Author(s):  
Sara Mantero ◽  
Federica Boschetti
Keyword(s):  
Culture Conditions ◽  
In Vitro Models ◽  
In Vivo Models ◽  
In Vitro Development ◽  
Culture Cells ◽  
Vitro Development ◽  
Tissue Models ◽  
Mechanical Stretching

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels.

Stage specific expression of cell cycle genes during in vivo or in vitro development of ovarian follicles in sheep

2016 ◽  
Vol 143 ◽  
pp. 1-7 ◽  
Author(s):  
V. Praveen Chakravarthi ◽  
S.S.R. Kona ◽  
A.V.N. Siva Kumar ◽  
M. Bhaskara ◽  
V.H. Rao
Keyword(s):  
Cell Cycle ◽  
Ovarian Follicles ◽  
Specific Expression ◽  
In Vitro Development ◽  
Cell Cycle Genes ◽  
Vitro Development

55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
F. Forell ◽  
J. C. Mezzalira ◽  
F. Zago ◽  
F. K. Vieira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Rate ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning ◽  
Activation Intervals

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

In vivo and in vitro development of in vitro fertilized bovine follicular oocytes obtained by laparoscopy

1985 ◽  
Vol 23 (1) ◽  
pp. 230 ◽  
Author(s):  
M.A. Sirard ◽  
R.D. Lambert ◽  
P. Guay ◽  
D.P. Ménard ◽  
M. Bedoya
Keyword(s):  
In Vitro Development ◽  
Vitro Development
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