Meiosis progression and donor age affect expression profile of DNA repair genes in bovine oocytes

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 11-18 ◽  
Author(s):  
S. Bilotto ◽  
R. Boni ◽  
G.L. Russo ◽  
M.B. Lioi
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Calf Serum ◽  
Internal Standard ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Gene Marker ◽  
Donor Age ◽  
Immature Oocytes

SummarySeveral genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are: (i) meiosis progression, from maturation to fertilization, due to extensive chromatin remodelling during genome decondensation; and (ii) aging, which is associated with a progressive oxidative stress. In this work, we studied the transcriptional patterns of three genes, RAD51, APEX-1 and MLH1, involved in DNA repair mechanisms. The analyses were performed by real-time quantitative PCR (RT-qPCR) in immature and in vitro matured oocytes collected from 17 ± 3-month-old heifers and 94 ± 20-month-old cows. Batches of 30–50 oocytes for each group (three replicates) were collected from ovarian follicles of slaughtered animals. The oocytes were freed from cumulus cells at the time of follicle removal, or after in vitro maturation (IVM) carried out in M199 supplemented with 10% fetal calf serum, 10 IU luteinising hormone (LH)/ml, 0.1 IU follicle-stimulating hormone (FSH)/ml and 1 μg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovine GAPDH gene was used as the internal standard, while primers for bovine RAD51, APEX-1 and MLH1 genes were designed from DNA sequences retrieved from GenBank. Results obtained indicate a clear up-regulation of RAD51, APEX-1 and MLH1 genes after IVM, ranging between two- and four-fold compared with germinal vesicle (GV) oocytes. However, only RAD51 showed a significant transcript increase between the immature oocytes collected from young or old individuals. This finding highlights RAD51 as a candidate gene marker for discriminating bovine immature oocytes in relation to the donor age.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

scholarly journals Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

2020 ◽  
pp. 1
Author(s):  
Tulay Irez ◽  
Sinem Ercan Dogan ◽  
Enver Ciraci ◽  
Saadet Busra Aksoyer ◽  
Muhammet Sait Toprak ◽  
...  
Keyword(s):  
Experimental Study ◽  
Luteinizing Hormone ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Immature Oocytes ◽  
Maturation Rate

<p><strong>OBJECTIVE:</strong> In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. </p><p><strong>STUDY DESIGN:</strong> In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. </p><p><strong>RESULTS:</strong> In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).</p><p><strong>CONCLUSION:</strong> In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.</p>

274 LIPOLYSIS IN CUMULUS CELLS ACCOMPANIES OOCYTE MATURATION IN BOVINE

2015 ◽  
Vol 27 (1) ◽  
pp. 226 ◽  
Author(s):  
S. Uzbekova ◽  
L. Sanchez-Lazo ◽  
A. Desmachais ◽  
V. Maillard ◽  
S. Elis
Keyword(s):  
Oocyte Maturation ◽  
Energy Homeostasis ◽  
Binding Proteins ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hormone Sensitive Lipase ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes

Oocyte maturation relies on energy from different nutrients, including fatty acids (FA). Cumulus cells (CC) are metabolically coupled with enclosed oocyte and active FA metabolism occurs in both compartments. Excess of lipids in oocyte environment alters its developmental competence. Lipid droplets (LD), mainly composed of triacylglycerides (TG), are formed inside of CC and in oocyte to store lipids. Liberation of free FA from TG requires lipolysis, which is catalyzed by lipases and involves FA-binding proteins (FABP) and perilipins (PLIN), which interact at the surface of LD as shown in lipogenic tissues. The objective was to elucidate the main factors involved in lipolysis in bovine cumulus-oocyte complex (COC) during oocyte maturation. Gene expression before and after maturation was analysed in CC by microarray hybridization and validated by real time RT-PCR; proteins were detected by Western blot and immunofluorescence. For statistics, ANOVA and Mann-Whitney (M-W) tests were used. In CC, adipose triglyceride lipase PNPLA2, lipoprotein lipase LPL, and monoacylglycerol lipase ABHD6 showed the highest mRNA expression level among 7 detected lipases. Both PLIN5 and PLIN2 were the most abundant perilipins, and among 8 FA-binding proteins, FABP3 and FABP5 were predominant. During in vitro maturation (IVM), expression of most of these genes increased at 6 h of IVM (P < 0.05, ANOVA) in CC. At that time, germinal vesicle breakdown occurred in enclosed oocytes and hyaluronan synthase HAS2, involved in the extra-cellular matrix formation, was upregulated in CC. The most upregulated genes after 18 h of IVM in CC were ABDH6 (48.5-fold as compared to immature, P < 0.01, M-W), FABP3 (16.6-fold, P < 0.01, M-W), and PLIN2 (5.5-fold, P < 0.05, M-W). Expression of all of these lipolysis-related genes was also detected in the oocytes. At the protein level, PLIN2 was mainly localised in the cytoplasmic LD, both in CC and in the oocyte. In CC, FABP3 was detected in the cytoplasm, whereas in oocyte it was also localised to the germinal vesicle of immature oocytes and closely to the chromosomes during the first meiotic division. In addition, active phosphorylated hormone sensitive lipase HSL was always detected in CC and in mature oocytes, but not in immature oocytes. All these data demonstrate that lipolysis occurs both in CC and in the oocyte during maturation. Lipolysis may be necessary to maintain cell energy homeostasis by regulating intracellular concentration of free FA. Moreover, CC were already described to store the excess FA from follicular fluid in order to protect the oocyte. Our data corroborate the essential role of CC in oocyte survival through controlling FA metabolism inside the COC. Active lipolysis may therefore be required to reduce lipid storages as well as to produce energy necessary for oocyte meiosis progression and extracellular matrix secretion by CC in order to prepare COC for further fertilization.This work was supported by INRA, ANR (OSCILE project) and European subvention FP7-KBBE-2012–6 (FECUND project).

OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Lun Suo ◽  
Guang-Bin Zhou ◽  
Qing-Gang Meng ◽  
Chang-Liang Yan ◽  
Zhi-Qiang Fan ◽  
...  
Keyword(s):  
Survival Rate ◽  
Cell Damage ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes

SummaryCryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

2021 ◽  
Author(s):  
Dulama Richani ◽  
Robert B Gilchrist
Keyword(s):  
Protein Synthesis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Protein Synthesis Inhibitors ◽  
Meiotic Arrest ◽  
Oocyte Developmental Competence

Abstract Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)?

1997 ◽  
Vol 9 (7) ◽  
pp. 703 ◽  
Author(s):  
A. H. Sathananthan ◽  
A. Szell ◽  
S. C. Ng ◽  
A. Kausche ◽  
O. Lacham-Kaplan ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Germinal Vesicle ◽  
Sperm Chromatin ◽  
Perivitelline Space ◽  
Chromatin Decondensation ◽  
Immature Oocytes ◽  
Human Oocytes ◽  
Ultrastructural Evidence ◽  
Intra Cytoplasmic Sperm Injection

There is debate as to whether the acrosome reaction is necessary for sperm incorporation after intra-cytoplasmic sperm injection (ICSI). Ultrastructural evidence is presented to show that the acrosome reaction could occur in the ooplasm before sperm incorporation in mature human oocytes or the acrosome could be discarded intact before sperm incorporation in immature oocytes, matured in vitro. Both germinal vesicle and growing follicular oocytes showed sperm chromatin decondensation, with discarded acrosomes close to the sites of incorporation, and were able to form male pronuclei. This is probably the first report of microfertilization of a growing oocyte with a reticulate nucleolus by ICSI. The acrosome reaction, when it occurs, is preceded by acrosome swelling and is followed by vesiculation of surface membranes exposing the inner acrosome membrane, as observed on the surface of the zona during IVF or in the perivitelline space after subzonal sperm injection. These sperm were probably capacitated at the time of ICSI. There was subtle evidence of leaching of the acrosomal matrix from intact discarded acrosomes and from partially depleted acrosomes attached to decondensing spermheads. These sperm were probably not fully capacitated at the time of ICSI. It is concluded that both the acrosome reaction and acrosome deletion are possible prerequisites to sperm incorporation after ICSI.

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

71 VITRIFICATION OF BOVINE OOCYTES IN MEDIA WITH GLYCEROL + ETHYLENE GLYCOL BEFORE AND AFTER IN VITRO MATURATION

2008 ◽  
Vol 20 (1) ◽  
pp. 116
Author(s):  
L. G. Devito ◽  
C. B. Fernandes ◽  
H. N. Ferreira ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Quiescent State ◽  
Developmental Potential ◽  
Immature Oocytes ◽  
Nitrogen Vapor ◽  
Bovine Oocytes

The cryopreservation process aims to keep the cellular metabolism in a quiescent state for an indeterminate length of time. In mammals, oocyte cryopreservation success is important for the establishment of genetic banks. The objective of the present experiment was to evaluate the effect of vitrification on oocyte meiotic ability and the integrity of the metaphase plate in immature and in vitro-matured bovine oocytes. Bovine cumulus–oocytes complexes (COCs) were harvested from slaughterhouse ovaries and randomly divided into 3 groups: (G1) non-vitrified oocytes subjected to in vitro maturation, (G2) immature oocytes vitrified and then subjected to in vitro maturation after warming, and (G3) in vitro-matured oocytes subjected to vitrification. For in vitro maturation, oocytes were incubated for 22 h in 5% CO2 in air in TCM-199 with fetal calf serum, estradiol, LH, FSH, pyruvate, and gentamicin. For vitrification, the oocytes were exposed to the cryoprotectors in three steps: solution 1 containing 1.4 m glycerol in PBS for five min, and then solution 2 containing 1.4 m glycerol and 3.6 m ethylene glycol in PBS for another five min. After exposure to the second solution, the oocytes were transferred to 30-µL drops of solution 3 containing 3.4 m glycerol and 4.6 m ethylene glycol, loaded (5 oocytes per straw) in less than 1 min into 0.25-mL straws between two columns of 0.5 m galactose in PBS separated by two air bubbles, and immediately set in liquid nitrogen vapor. After 1 min of equilibration in liquid nitrogen vapor, the straws were immersed in liquid nitrogen. Warming was performed by holding the straws for 10 s in air, followed by 10 more s in a water bath at 20–22�C. The straws were then shaken 5 to 8 times to mix the bubbles (movement similar to that for a thermometer) and left horizontally for 6 to 8 min at room temperature. The rates of metaphase II and degeneration were analyzed by ANOVA followed by the Student t-test. The oocytes were stained with 100 µg mL–1 Hoechst 33342 and examined in an inverted microscope equipped with fluorescent light (UV filters 535 and 617 mm). Three different routines were realized with a total of 90 oocytes per group. The metaphase II rates in G1 (48/90, 53.3%) and G3 (42/90, 46.6%) were statistically the same (P e 0.05), but were higher (P d 0.05) than in G2 (0/90, 0%). The degeneration rates were: G1 (18/90, 20%), G2 (77/90, 85.6%), and G3 (7/90, 7.8%). The vitrification procedure damaged mainly the immature oocytes, since in the G2 the degeneration rate was higher and the oocytes were not able to resume meiosis. Meanwhile, when oocytes were vitrified after in vitro maturation, the metaphase II rate was similar to the one observed in IVM oocytes not subjected to vitrification. This indicates that the vitrification procedure performed in this experiment did not damage the structure of the metaphase II plate. However, more studies are necessary to predict the developmental potential of these in vitro-matured oocytes.

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

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