354 POOR EMBRYO DEVELOPMENT AFTER ICSI WITH DOMESTIC CAT TESTICULAR SPERM IS OVERCOME BY CENTROSOME AND MIDPIECE REPLACEMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 284
Author(s):  
P. Comizzoli ◽  
D. Wildt ◽  
B. Pukazhenthi
Keyword(s):  
Cell Cycle ◽  
Embryo Development ◽  
Polar Body ◽  
Domestic Cat ◽  
Testicular Sperm ◽  
Developmental Potential ◽  
Kinetics Of ◽  
Sperm Aster ◽  
Fisher Scientific

Testicular sperm from the cat can be used via intracytoplasmic sperm injections (ICSI) to produce embryos in vitro, but the proportion of morulae and blastocysts is less than that obtained using ejaculated sperm. Compromised embryo development has been linked to the inability of the cat testicular sperm centrosome to form a normal sperm aster during the first cell cycle post-ICSI. The aim of the present study was to improve embryo development after ICSI with a testicular spermatozoon by centrosome/midpiece replacement from an ejaculated spermatozoon. Sperm suspensions used for ICSI (fresh testicular sperm from one adult testis vs. frozen-thawed sperm from one ejaculate in each replicate; four replicates) were sonicated for 3 s at setting 3 using a 60 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, USA) to separate heads from centrosome/midpieces. Control immunostaining with monoclonal centrin antibodies revealed that centrosomes were attached to midpieces and were separated from sperm heads after sonication. A single sperm head (testicular, Th; ejaculated, Eh) was carefully positioned proximal to a single centrosome/midpiece (testicular, Tc; ejaculated, Ec) before injection into an oocyte with a visible polar body (in each replicate, n = 18 oocytes injected with each ThEc, EhEc, EhTc, or ThTc combination; four replicates). Injected oocytes then were activated with 7% ethanol and cultured in vitro in Ham's F10 (38.5�C, 5% CO2 in air). Percentages of first cleavages were recorded from 20 to 32 h post-activation (hpa). Nuclear status of uncleaved oocytes was evaluated at 48 hpa, and embryo development was assessed after 7 days of in vitro culture. Values were expressed as mean � standard deviation and analyzed by ANOVA. None of the uncleaved oocytes were activated. Percentages of cleaved oocytes (relative to the total number of injected oocytes) were not different (range, 61-65%; P > 0.05) among combinations of sperm heads and centrosome/midpieces. The mean times of the first cleavage, however, were earlier (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 25.1 � 1.1 h; EhEc, 25.0 � 0.9 h) than with testicular centrosome/midpieces (EhTc, 29.2 � 0.8 h; ThTc, 29.3 � 1.2 h). Percentages of morulae produced were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 15.3 � 2.7%; EhEc, 16.7 � 2.1%) than with testicular centrosome/midpieces (EhTc, 8.3 � 3.1%; ThTc, 6.9 � 2.2%). Likewise, percentages of blastocysts were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 12.5 � 3.0%; EhEc, 13.9 � 2.8%) than with testicular centrosome/midpieces (EhTc, 5.6 � 3.3%; ThTc, 6.9 � 2.9%). These results demonstrated that: (1) kinetics of the first cell cycle and the success of embryonic development were determined by centrosome/midpiece source; and (2) poor developmental potential of domestic cat testicular sperm could be circumvented by centrosome/midpiece replacement from an ejaculated spermatozoon.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

Developmental potential and kinetics of pig embryos with different cytoplasmic volume

Zygote ◽  
2013 ◽  
Vol 23 (2) ◽  
pp. 277-287 ◽  
Author(s):  
Juan Li ◽  
Rong Li ◽  
Klaus Villemoes ◽  
Ying Liu ◽  
Stig Purup ◽  
...  
Keyword(s):  
Embryo Development ◽  
Common Pattern ◽  
Developmental Potential ◽  
Cell Cycles ◽  
Cell Numbers ◽  
Reconstructed Embryos ◽  
Kinetics Of

SummaryThe effects of cytoplasmic volumes on development and developmental kinetics of in vitro produced porcine embryos were investigated. During hand-made cloning (HMC), selected cytoplasts were separated into two groups according to their size in relation to the initial oocyte: ~75% or ~50%. Following two fusion steps and activation (day 0), reconstructed embryos were cultured in vitro for 6 days. Cleavage rates on day 2 as well as blastocyst rates and cell numbers on day 6 were recorded. Results showed that embryo development was no different for ~50% versus ~75% cytoplasm at first fusion. This result was used in the following experiments, where the effect of varying cytoplasm volume in second fusion to obtain a final cytoplasm volume of ~75% to ~200% was tested. The results showed that the lowest quality was obtained when the final cytoplasm volume was ~75% and the highest quality at ~200% of the original oocyte. Similar results were observed in parthenogenetic (PA) embryos activated with different cytoplasmic volumes. A common pattern for the developmental kinetics of HMC and PA embryos was observed: the smaller group tended to have a longer time for the first two cell cycles, but subsequently a shorter time to form morula and blastocyst. In conclusion, the developmental kinetics of in vitro produced embryos was affected by the cytoplasm volume of the initial oocyte, and this further accounted for the developmental ability of the reconstructed embryos.

scholarly journals Effects of suramin on cell-cycle kinetics of MCF-7 human breast cancer cells in vitro

1993 ◽  
Vol 67 (2) ◽  
pp. 232-236 ◽  
Author(s):  
JA Foekens ◽  
AM Sieuwerts ◽  
EMJ Stuurman-Smeets ◽  
HA Peters ◽  
JGM Klijn
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Human Breast Cancer ◽  
Breast Cancer Cells ◽  
Human Breast Cancer Cells ◽  
Human Breast ◽  
Cell Cycle Kinetics ◽  
Kinetics Of ◽  
Mcf 7

scholarly journals Mitochondria and human preimplantation embryo development

Reproduction ◽  
2009 ◽  
Vol 137 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Martin Wilding ◽  
Gianfranco Coppola ◽  
Brian Dale ◽  
Loredana Di Matteo
Keyword(s):  
Embryo Development ◽  
Preimplantation Embryo ◽  
Anaerobic Respiration ◽  
Mitochondrial Metabolism ◽  
Human Reproduction ◽  
Aerobic Respiration ◽  
Human Embryos ◽  
Developmental Potential ◽  
Preimplantation Embryo Development

Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

341 OXYGEN TENSION AND EGF AFFECT METABOLISM OF IN VITRO-MATURED CUMULUS-ENCLOSED MOUSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 278
Author(s):  
K. A. Preis ◽  
G. E. Seidel Jr ◽  
D. K. Gardner
Keyword(s):  
Glucose Uptake ◽  
Oxygen Tension ◽  
Embryo Development ◽  
Metabolic Activity ◽  
Lactate Production ◽  
Defined Medium ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Exact Test

In vitro maturation of immature oocytes results in limited success in both clinical and research laboratories. Although reduced oxygen concentration is beneficial to embryo development, the optimal concentration for oocyte maturation has yet to be determined. The objective of this study was to determine whether oxygen tension (20% or 5% O2) affects oocyte physiology. Additionally, the effect of epidermal growth factor (EGF) in maturation medium on oocyte metabolic activity and subsequent embryo development was determined. Cumulus–oocyte complexes (COCs; n = 231) were collected from 28-day-old unprimed F1 (C57BL/6 × CBA/ca) mice. COCs were individually matured in defined medium at 37°C in 6% CO2 in one of four groups (Table 1). For the metabolism study, COCs were further divided into two groups: individual maturation in a 2-µL drop of medium for 16 h (n = 131); or individual maturation in 5-μL for 12 h and then placed in a 0.5-μL drop of medium for 4 h (n = 100), the time of greatest metabolic activity of the COC. At 17 h of maturation, COCs were individually fertilized, and zygotes were individually cultured until 96 h, at which time blastocyst development was assessed. Metabolic profiles were analyzed by ANOVA, and blastocyst rates were analyzed by Fisher's exact test. Maturation rates and blastocyst development were not different between groups. However, at 12–16 h of maturation, metabolism of COCs was affected by both oxygen tension and EGF (Table 1). Concerning metabolism over the entire course of maturation, glucose uptake and lactate production were higher in COCs in 5% O2 + 100 ng EGF (P < 0.05) than in the remaining three groups. There was no difference between 5% O2 and 20% O2 + 100 ng EGF, but 20% O2 caused less glucose uptake and lactate production than did the other three treatment groups (P < 0.05). Results of this study are the first to show that oxygen tension alters COC metabolism: COCs matured under 5% O2 were more active metabolically than COCs matured under 20% O2. The effect of oxygen tension is to some extent moderated by the presence of EGF, as metabolic activity of COCs matured under 20% O2 + 100 ng EGF was closer to that of COCs matured under 5% O2 conditions. Although blastocyst rates were similar across the four groups, embryos derived from oocytes matured in different oxygen tensions may exhibit different developmental potential. In conclusion, results of this study have implications for the improvement of maturation conditions in both clinical and research laboratories. Table 1. Carbohydrate metabolism of individual COCs at 12–16 h of maturation

197 INHIBITION OF CYSTEINE PROTEINASES DURING IN VITRO OOCYTE MATURATION IMPROVES DEVELOPMENT OF PORCINE CUMULUS - OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 211
Author(s):  
A. M. Lichtenauer ◽  
L. D. Spate ◽  
R. S. Prather ◽  
J. A. Green
Keyword(s):  
Oocyte Maturation ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Developmental Potential

Biochemical differences exist between oocytes that give rise to viable blastocysts and oocytes that give rise to embryos that are developmentally compromised. For example, specific proteolytic enzymes (e.g. cathepsin B) are transcriptionally abundant in in vitro-matured bovine oocytes from prepubertal heifers that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus–oocyte complexes (COC) that represented both poor- and good-quality oocytes. Those reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development. This project sought to determine if similar results would be obtained in a porcine oocyte/embryo culture system. Inclusion of 10 and 20 μM E-64 in maturation medium was performed. Maturation rates of porcine COC in 20 μM E-64 were elevated compared to those incubated in 10 μM E-64 (74% vs 53%; P < 0.05) or without E-64 (55%; P < 0.05: N = 1750 oocytes tested). Successful maturation to metaphase II was based on the presence of a polar body and a uniform cytoplasm 44 h after follicular aspiration. Based on these preliminary results and the earlier bovine work, it was hypothesized that the E-64 was having little influence on normal oocytes, but was promoting maturation of low-quality oocytes, possibly those that were beginning to degenerate. Consequently, 20 μM of E-64 was added to the maturation media of COC segregated based on morphological characteristics of the oocytes. Good COC had a homogeneous cytoplasm and greater than 3 layers of cumulus cells; the COC were considered poor if they displayed a nonhomogeneous cytoplasm and 1 layer or less of cumulus cells, yet were still considered fertilizable. Without E-64, an increase in maturation was measured when good oocytes were compared to poor oocytes (52% vs 29%; P < 0.05: N = 1600). No significant differences in maturation were observed between good oocytes incubated in the presence or absence of E-64. Likewise, no significant differences were observed between poor oocytes incubated in the presence or absence of E-64. The percentage of maturation of good oocytes cultured in E-64 was significantly higher than that of poor oocytes cultured with E-64 (67% vs 43%; P < 0.05). Maturation with the inhibitor did not significantly affect the subsequent cleavage or blastocyst rates of embryos that arose from these oocyte groups after fertilization. These experiments suggest that inhibition of cysteine proteinases significantly promotes oocyte maturation, as was seen in previous bovine work. Our data did not support the hypothesis that cysteine proteinase inhibition was selectively improving maturation of poor oocytes within the pool. It remains possible that increased maturation in good oocytes is a result of cysteine inhibition on juvenile oocytes that morphologically appeared good and the effect was less on already degenerated oocytes that appeared poor. Differences between treatments were determined by ANOVA with post-test by Tukey's multiple comparison test.

53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
R. Olivera ◽  
C. Alvarez ◽  
I. Stumpo ◽  
G. Vichera
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Nuclear Reprogramming ◽  
Chi Square ◽  
In Vitro Development ◽  
First Polar Body ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

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