15 EFFECTS OF DEMECOLCINE ON THE MEIOTIC CELL-CYCLE AND MICROTUBULAR KINETICS OF ACTIVATED BOVINE OOCYTES SUBMITTED TO CHEMICAL ENUCLEATION

2012 ◽  
Vol 24 (1) ◽  
pp. 119
Author(s):  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
T. A. D. Tetzner ◽  
M. R. de Lima ◽  
S. C. Méo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Time Course ◽  
Polar Body ◽  
Invasive Procedure ◽  
Activation Process ◽  
Negative Consequences ◽  
Free Medium ◽  
Significance Level ◽  
Bovine Oocytes

Enucleation in traditional nuclear transfer (NT) is an invasive procedure for which alternative protocols have been sought. The present study was designed to explore the time course effects of demecolcine, a microtubule-depolymerizing agent, in bovine-activated oocytes submitted to induced chemical enucleation. For that purpose, after 26 h of in vitro maturation, the oocytes were parthenogenetically activated (5 μM ionomycin for 5 min and 10 μg mL–1 cycloheximide for 4 h) and treated with demecolcine (0.05 μg mL–1 for 2 h) 2 h after activation. Three groups were established: control (untreated oocytes), activated (oocytes exposed to activation) and deme (oocytes activated and treated with demecolcine). Then the nuclear and microtubular dynamics of the oocytes were evaluated by immunofluorescence microscopy of tubulin and chromatin (Liu et al. 1998 Biol. Reprod. 59, 537–545). In each one of 3 replicates, 15 to 30 oocytes were evaluated per group. Oocytes were classified according to microtubule (MT) patterns as follows: present (evident MT), reduced (MT with reduced density), or absent. The results in percentage were submitted to ANOVA and means were compared by Tukey test, with a significance level of 5%. Effects of activation were observed after 2 h, when higher rates of oocytes presenting second polar body (2nd PB) extrusion were observed in the groups activated and deme (49.3% in both groups) compared with control (1.7%). At the end of activation treatment (4 h), the activated group presented 81.8% of oocytes with 2nd PB extrusion, whereas it was observed only in 37.8% of oocytes in the deme group. Effects of demecolcine on the microtubules initiated after only 0.5 h of treatment, when an increase (P < 0.05) of oocytes with reduced MT was observed in the deme group (26%; control – 3%; activated – 0%). After 6 h of culture in demecolcine-free medium, the deme group displayed ∼50% of oocytes with reduced MT (control – 0%; activated – 39%) and absence of MT in 23% of oocytes, which was superior to other groups (control – 0%; activated – 2%). Therefore, we detected a reduction of MT density after exposition of activated oocytes to demecolcine. However, MT were not completely absent in most of the evaluated oocytes, as previously described for bovine oocytes submitted to chemically assisted enucleation (Saraiva et al. 2009 Cloning Stem Cells 11, 141–152; Meng et al. 2011 Cell. Reprogram., in press). Apparently, there was no immediate repolymerization of MT after culture in demecolcine-free medium and this could result in negative consequences for subsequent embryo development. Moreover, demecolcine impaired the second PB extrusion during the activation process, probably due to inhibition of spindle rotation caused by the MT-disrupting drug. Nonetheless, considering the higher cytoplasmatic volume obtained with chemical enucleation and the lesser extent of injuries suffered by recipient oocytes, further studies focusing on the potential of embryo development and the quality of embryos are advisable. Financial support: FAPESP.

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

188 HAPLOID ACTIVATION OF BOVINE OOCYTES WITH IONOMYCIN AND SINGLE OR COMBINED ACTIVATING AGENTS

2016 ◽  
Vol 28 (2) ◽  
pp. 225 ◽  
Author(s):  
M. Suvá ◽  
N. G. Canel ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Reproductive Technologies ◽  
Mitogen Activated Protein Kinase ◽  
Cleavage Rate ◽  
Extrusion Rate ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Bovine Oocytes

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P < 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation. Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1

Selection of bovine oocytes by brilliant cresyl blue staining: effect on meiosis progression, organelle distribution and embryo development

Zygote ◽  
2011 ◽  
Vol 21 (3) ◽  
pp. 250-255 ◽  
Author(s):  
D.S. Silva ◽  
P. Rodriguez ◽  
A. Galuppo ◽  
N.S. Arruda ◽  
J.L. Rodrigues
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Blue Staining ◽  
Cortical Granule ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Cytoplasmic Maturation ◽  
Bovine Oocytes ◽  
Selection Of

SummaryThe selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 μM BCB stain and classified according to the aspect of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB− (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p < 0.05) higher in BCB+ oocytes than in BCB− (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB+ and BCB− groups, while blastocyst rates on D7 were (p < 0.05) higher (35%) in BCB+ vs BCB− (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

The effect of temperature during liquid storage of in vitro–matured bovine oocytes on subsequent embryo development

2016 ◽  
Vol 85 (3) ◽  
pp. 509-518.e1 ◽  
Author(s):  
Tayita Suttirojpattana ◽  
Tamas Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Effect Of Temperature ◽  
Liquid Storage ◽  
Bovine Oocytes

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 69-76 ◽  
Author(s):  
S.A. Ock ◽  
J.S. Bhak ◽  
S. Balasubramanian ◽  
H.J. Lee ◽  
S.Y. Choe ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chromosomal Abnormality ◽  
Polar Body ◽  
Ultrastructural Changes ◽  
Time Interval ◽  
Oocyte Activation ◽  
Group 4 ◽  
Group 5 ◽  
Bovine Oocytes

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 μM ionomycin for 5 min (group 3), 5 μM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 µM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

scholarly journals Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 861-874 ◽  
Author(s):  
Korakot Nganvongpanit ◽  
Heike Müller ◽  
Franca Rings ◽  
Michael Hoelker ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Free Water ◽  
Double Stranded Rna ◽  
Immature Oocytes ◽  
Selective Degradation ◽  
First Polar Body ◽  
Bovine Oocytes

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2,in vitroproduced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

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.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

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