Study on Isolation of Inner Cell Mass from Bovine Embryo Derived from In Vitro Fertilization by Exposure to Calcium Ionophore A23187

Mouse inner cell masses remained intact when exposed to extreme bsmotic stress (distilled water) for short periods, but the trophectoderm was lysed in one-third of blastocysts. However, these inner cell masses were not viable as they could not fluoresce after incubation in fluorescein diacetate nor continue development in vitro. It was concluded that cells of the inner cell mass are not more tolerant of osmotic stresses than those of the trophectoderm.

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Isolation and development of the inner cell mass after exposure of mouse embryos to calcium ionophore A23187

Development ◽

10.1242/dev.45.1.237 ◽

1978 ◽

Vol 45 (1) ◽

pp. 237-247

Author(s):

M. Azim H. Surani ◽

David Torchiana ◽

Sheila C. Barton

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Embryoid Bodies ◽

Detectable Effect ◽

Ionophore A23187 ◽

Culture Dish

Compacted morulae and blastocysts were obtained from CBA, BALB/c and CFLP strains of mice. The embryos were incubated in medium containing 2 × 10−5 M or 2 × 10−6 M ionophore A 23187. With 2 × 10−6 M ionophore, morulae survived for up to 12 h showing slight decompaction. Normal development resumed when the morulae were explanted to fresh medium. There was no detectable effect on blastocysts. With 2 × 10−5 M ionophore, morulae survived for about 20 min and then extensive cell death occurred after this time. With blastocysts however, selective lysis of trophectoderm cells occurred after approximately 30 min following their swelling and vesiculation but the inner cell mass cells (ICM) remained apparently intact and viable. Nearly 80% of the early blastocysts obtained 87 h postovulation and all of the late blastocysts used after 12 h in culture (99 h blastocysts) showed this response. Individual fluid accumulating cells were detected in a few isolated ICMs after their overnight culture in vitro, especially in those obtained from early blastocysts, but the majority of the ICMs did not have these cells. All aggregates of three to five ICMs, except one which reformed into a blastocyst, developed as embryoid bodies after 2 days in culture and these survived for up to 10 days; in some cases they developed into cystic embryoid bodies or attached to the culture dish displaying a variety of cell types. The development of the isolated ICMs in vivo was judged to be normal after their transfer to intact host blastocysts as these developed as chimaeric embryos to term.

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In vitro fertilization and culture alters chromatin accessibility in the mouse inner cell mass

Fertility and Sterility ◽

10.1016/j.fertnstert.2018.07.1056 ◽

2018 ◽

Vol 110 (4) ◽

pp. e378

Author(s):

E. Ruggeri ◽

E. Grow ◽

X. Liu ◽

A. Donjacour ◽

P. Rinaudo

Keyword(s):

In Vitro Fertilization ◽

Inner Cell Mass ◽

Cell Mass ◽

Chromatin Accessibility ◽

Vitro Fertilization

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Effects of Trophectoderm Dissection on Development of Inner Cell Mass of Fresh and Frozen-Thawed Bovine Blastocysts Derived from In-Vitro Fertilization.

Journal of Reproduction and Development ◽

10.1262/jrd.39.281 ◽

1993 ◽

Vol 39 (4) ◽

pp. 281-286

Author(s):

Xihe LI ◽

Setsuo IWASAKI ◽

Tatsuo NAKAHARA

Keyword(s):

In Vitro Fertilization ◽

Inner Cell Mass ◽

Cell Mass ◽

Vitro Fertilization

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250 DISTRIBUTION OF HISTONE macroH2A1 IN BOVINE PRE-IMPLANTATION EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab250 ◽

2006 ◽

Vol 18 (2) ◽

pp. 232

Author(s):

C. Kim ◽

Y. Ma ◽

C.-C. Chang ◽

T. Rasmussen ◽

X. Yang ◽

...

Keyword(s):

In Vitro Fertilization ◽

Laser Scanning ◽

Expression Patterns ◽

Combined Treatment ◽

Calcium Ionophore ◽

Ionophore A23187 ◽

Bovine Embryos ◽

Vitro Fertilization ◽

Stage Of Development

It is known that heterochromatin is characterized by the presence of the histone variant of macroH2A1. MacroH2A1 is a core variant histone with a hybrid structure consisting of a domain that resembles a full-length histone H2A1 followed by a large nonhistone domain. We have previously studied the dynamic changes of macroH2A1 accumulation during the pre-implantation developmental period in the mouse. In the present study, we investigated the distribution of microH2A1 in bovine metaphase II oocytes and pre-implantation embryos at 2-, 4-, 8-, 16-cell, and morula stages as well as blastocysts harvested at Days 8, 9, 10, 11, 12, and 13 following activation and in vitro fertilization (IVF). To generate parthenotes, denuded and in vitro-matured oocytes were activated using a combined treatment of calcium ionophore A23187, cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP). Five oocytes and pre-implantation embryos at each stage of development were used to follow the development expression pattern of microH2A1 by immunocytochemistry. The cross-reactivity of the primary antibody against mouse microH2A1 was verified by Western blot analysis with bovine fibroblasts. Another staining control included immunostaining with antibody against histone molecules. The stained embryos were observed by laser-scanning confocal microscopy and epiflourescence microscopy. No microH2A1 stain was observed in bovine oocytes or pre-implantation embryos up to the expanded blastocyst stages. In the IVF group, the macroH2A1 was first found in elongated blastocysts (Day 11) after hatching. We observed different expression patterns of macroH2A1 in activated vs. IVF bovine embryos. In the parthenote group, we failed to find robust expression even when embryos were cultured for 13 days. Moreover, the pattern of macroH2A1 expression in bovine embryos was different fromn that in the mouse, in which the onset of macroH2A1 expression occurred by the 16-cell morula stage. These results suggest species differences in the establishment of epigenetic signals. This work was supported by grants from USDA to X. Y. and X. C. T.

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139 Effect of protein and calcium ionophore A23187 concentration on hyperactivated motility and acrosome status of stallion sperm

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab139 ◽

2019 ◽

Vol 31 (1) ◽

pp. 195

Author(s):

I. Ortiz ◽

H. Resende ◽

M. Felix ◽

C. Love ◽

K. Hinrichs

Keyword(s):

Repeated Measures ◽

Calcium Influx ◽

Semen Analysis ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Computer Assisted ◽

Ionophore A23187 ◽

Variable Effect ◽

Vitro Fertilization

In vitro fertilization does not occur readily in the horse. Fertilization can be achieved using sperm treated with the calcium ionophore A23187 (CaI), but rates are low and variable. In order to fertilize, it is thought that the sperm must show hyperactivated motility and undergo the acrosome reaction. The presence of protein in the media is thought to suppress the effect of CaI, but protein is needed for maintenance of sperm motility. Therefore, the objective of this study was to assess the effect of CaI in the presence or absence of protein on the acrosome and on hyperactivated motility of equine sperm. For this purpose, sperm from 4 stallions were exposed for 10min at 37°C to vehicle or to 1 (C1), 5 (C5) or 10 (C10) μM CaI, with (BSA) or without (N) 7mg mL−1 BSA. The sperm were then washed and incubated at 37°C for 2h. Total and hyperactivated motility were measured by computer-assisted semen analysis. Sperm were considered hyperactivated if curvilinear velocity was >180μm s−1, amplitude of lateral head displacement was >12μm, linearity was <30% and fractal dimension value was >1.3. The percentage of live acrosome-reacted sperm was measured by flow cytometry after staining with propidium iodide and Pisum sativum agglutinin. Data were analysed by repeated-measures 2-way ANOVA. Results were expressed as mean±standard error. Total motility in C5 and C10 treatments was significantly decreased in relation to control (BSA-vehicle) starting at 30min of incubation (35.42±13.57 to 28.20±13.10% v. 71.72±9.21%, respectively; P<0.05). Hyperactivated motility was significantly lower in C10, C5 and N-C1 than in control after 2h of incubation (1.46±0.64v. 3.10±0.58%, respectively). Live acrosome-reacted sperm were significantly higher (P<0.05) for BSA-C5 (14.04±1.99%) and BSA-C10 (14.85±2.52%) than for control (7.50±1.62%) after 2h of incubation. The exposure to sperm of concentrations ≥5μM CaI was associated with loss of motility from 30min of incubation on. However, 2h of incubation after ≥5 μM CaI in the presence of BSA were needed to increase the percentage of live acrosome-reacted sperm. This mismatch between motility and acrosome response helps to clarify the reasons for the variable effect of sperm CaI treatment on equine IVF. Further studies measuring calcium influx and assessing the effect of sperm pre-incubation on CaI response are needed to explore mechanisms for equine in vitro sperm capacitation.

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Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro

Reproduction Fertility and Development ◽

10.1071/rd01020 ◽

2001 ◽

Vol 13 (6) ◽

pp. 383 ◽

Cited By ~ 8

Author(s):

Jin-Tae Chung ◽

Bruce R. Downey ◽

Robert F. Casper ◽

Ri-Cheng Chian

Keyword(s):

Embryonic Development ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Early Embryonic Development ◽

Ionophore A23187 ◽

Vitro Fertilization ◽

Parthenogenetic Activation ◽

Treatment Groups ◽

Bovine Oocytes

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL–1 FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen–thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 M calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v. 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (10.8% v. 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca2+-ionophore. However, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10 000g and stimulation with calcium ionophore A23187 than in the control (18.4% v. 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be ≤7000g to enhance the visibility of nuclear elements for further micromanipulation.

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205 CAPACITATION OF STALLION SPERMATOZOA EVALUATED BY FERTILIZATION OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab205 ◽

2008 ◽

Vol 20 (1) ◽

pp. 181

Author(s):

M. R. Hudson ◽

G. E. Seidel Jr ◽

E. L. Squires ◽

B. E. Spizzirri ◽

D. J. Walker ◽

...

Keyword(s):

Calcium Ionophore ◽

Cumulus Cells ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Dependent Manner ◽

Cell Stage ◽

Vitro Fertilization ◽

Cell Divisions ◽

Bovine Oocytes

In vitro fertilization in the horse does not work reliably. Several methods of capacitating sperm in other species fail in the horse. The goal of this experiment was to develop a method to capacitate equine spermatozoa using calcium ionophore A23187 or phosphatidylcholine 12 (PC12). We also studied effects of maturing bovine oocytes for 24 or 28 h on fertilizability by capacitated equine sperm, hypothesizing that longer maturation would yield oocytes more easily fertilized by equine spermatozoa. Two sets of bovine oocytes were aspirated from 3 to 8 mm follicles of abattoir ovaries 4 h apart, but fertilized at the same time. On the day of fertilization, semen from 1 of 3 stallions was collected, evaluated, and centrifuged through 33% Percoll to remove seminal plasma. The resultant pellet was extended to 5 × 107 cells mL–1 in M199 containing 0.6% BSA, 2 mm caffeine, and 5 mm CaCl2. Sperm were treated with A23187 (1 or 3 μm) or PC12 (40 or 70 μm) or both A23187 and PC12 (1 μm/40 μm) in 500- μL aliquots. Sperm were incubated at 39°C for 10 min (for A23187 and combination treatments) or 15 min (for PC12 treatments), and then diluted 1:20 for fertilization. Oocytes from each maturation time were fertilized using the same semen preparation for each treatment. Oocytes and sperm were incubated together for 18 h in FCDM in 5% CO2 at 39°C (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596). Presumptive zygotes were cultured for 30 h in CDM-1, vortexed to remove cumulus cells, and evaluated for cleavage. Oocytes were also co-incubated with killed sperm to determine the level of parthenogenesis. Cleaved embryos were stained with orcein to ensure that each cell had a nucleus. Number of cell divisions were recorded as 0 for a 1-cell, 1 for a 2-cell, 1.5 for a 3-cell, etc. More oocytes cleaved after 28 h (18%) than 24 h (14%) maturation (P < 0.01). Sperm of Stallion 1 resulted in higher overall cleavage (24%) than Stallions 2 or 3 (11 and 12%; P < 0.01). Highest cleavage was seen with 28 h maturation and 70 μm PC12 and 3 μm A23187 (27 and 24%, respectively). The most cell divisions were seen with 28 h maturation and 70 μm PC12 (0.48); 28 of the 49 cleaved in this treatment reached ≥4-cell stage. In conclusion, both A23187 and PC12 were able to capacitate equine sperm in a dose-dependent manner as determined from cleavage of bovine oocytes matured for 28 h; maturation for the conventional 24 h was an inferior model for this purpose. Table 1. Mean responses of bovine oocytes fertilized by equine sperm

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