25 Effect of roscovitine on the cumulus cells expansion, oocyte maturation and in vitro development of domestic cat embryos generated by in vitro fertilisation

2022 ◽  
Vol 34 (2) ◽  
pp. 246
Author(s):  
D. Veraguas-Davila ◽  
D. Saez-Ruiz ◽  
M. C. Alvarez ◽  
F. Saravia ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
In Vitro Fertilisation ◽  
In Vitro Development ◽  
Vitro Development

212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

scholarly journals Effect of granulosa and cumulus cells on in vitro development of the bovine follicular oocytes

1995 ◽  
Vol 8 (4) ◽  
pp. 317-320
Author(s):  
K. S. Im ◽  
H. J. Kim ◽  
K. M. Chung ◽  
H. S. Kim ◽  
K. W. Park ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
In Vitro Development ◽  
Vitro Development

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

In vitro development of morulae from immature caprine oocytes

Zygote ◽  
1994 ◽  
Vol 2 (2) ◽  
pp. 97-102 ◽  
Author(s):  
Levent Keskintepe ◽  
Gamal M. Darwish ◽  
Abdelmoneim I. Younis ◽  
Benjamin G. Brackett
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Glycoprotein Hormones ◽  
Glycoprotein Hormone ◽  
In Vitro Development ◽  
Morula Stage ◽  
Vitro Development ◽  
Hormone Control ◽  
Medium Supplementation

SummaryThe effects of medium supplementation with oestrous goat serum and glycoprotein hormones on caprine oocyte maturation in vitro (IVM) were evidenced by proportions of resulting ova completing in vitro fertilisation (IVF) and development to the morula stage. Oocyte-cumulus complexes (OCCs) were harvested in follicular fluid from 2–5 mm diameter follicles. Oocyte maturation took place during 27 h in TCM-199 supplemented with 20% oestrous goat serum, oestradiol-17β (1.0 μg/ml), and either (a) 0.5 μg FSH/ml, (b) 100 μg LH/ml, (c) 100 μg LH + 0.5 μg FSH/ml, (d) 100 μg hCG + 0.5 μg FSH/ml, (e) 0.5 μg TSH/ml or (f) no added glycoprotein hormone (control). Of 353 immature oocytes cultured in seven experiments, 311 (88.1%) exhibited cumulus expansion at the end of the IVM interval; all normalappearing OCCs were inseminated. In vitro insemination was with ejaculated sperm treated with heparin (10 μg/ml) and caffeine (0.4 μg/ml). Proportions (%) of inseminated ova that were fertilised (cleaved) and that reached the morula stage after IVM with (a) FSH, (b) LH, (c) LH + FSH, (d) hCG + FSH, (e) TSH and (f) no added glycoprotein hormone were (a) 22/52 (42.3%) and 9/52 (17.3%), (b) 25/54 (46.3%) and 14/54 (25.9%), (c) 52/65 (80.0%) and 26/65 (40.0%), (d) 48/78 (61.5%) and 22/78 (28.2%), (e) 14/54 (25.9%) and 4/54 (7.4%), and (f) 11/50 (22.0%) and 1/50 (2.0%), respectively. All treatments yielded better results than IVM with no added glycoprotein hormone. After IVM with added LH + FSH higher proportions of oocytes were fertilised (p<0.05), and higher proportions reached the morula stage (p<0.05) when compared with other treatments.

294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 262
Author(s):  
W. Fujii ◽  
H. Funahashi
Keyword(s):  
Chemical Activation ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Female Rats ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Pronuclear Formation ◽  
Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 142
Author(s):  
J. Galiguis ◽  
M. C. Gómez ◽  
C. E. Pope ◽  
B. L. Dresser ◽  
S. P. Leibo
Keyword(s):  
Lipid Content ◽  
High Speed ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Chi Square ◽  
In Vitro Development ◽  
Intracellular Lipids ◽  
Fetal Fibroblasts ◽  
Vitro Development

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca2+, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca2+ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca2+ (P < 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P < 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P < 0.05), 38 (P < 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P < 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca2+-free medium containing 10% FBS and 20.0 μg mL–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.

75 EPIGENETIC EFFECTS OF VITRIFICATION ON PRONUCLEAR DOMESTIC CAT EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 131 ◽  
Author(s):  
J. H. Galiguis ◽  
C. E. Pope ◽  
M. N. Biancardi ◽  
C. Dumas ◽  
G. Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genetic Material ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Epigenetic Effects ◽  
Donor Cells ◽  
Vitro Development

Vitrification remains a promising technique in the preservation of valuable genetic material; however, in the cat, success has varied. Live kittens have been produced from embryos vitrified at early cleavage stages, but phenotypic abnormalities in some kittens suggest possible epigenetic effects of the vitrification process. It has been reported that cryopreservation alters epigenetic events in somatic donor cells, which indirectly influences physical status of cloned offspring. However, extending post-warming in vitro culture of donor cells corrects these epigenetic modifications, resulting in normal embryos/clones. Accordingly, in the present study, vitrification was performed at the pronuclear stage to lengthen pretransfer culture time, and vitrified cat zygotes were assessed by analysing (1) histone acetylation/methylation, (2) global DNA methylation, (3) pluripotent gene expression, (4) in vitro development, and () in vivo viability. In vivo matured/IVF oocytes were vitrified in 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose at 16 h post-insemination (PI). After warming in 1.0 M sucrose at 38°C, embryos were fixed at 18 h or 40 h PI, and the nuclear intensity of either acetyl/dimethyl-H3K9 or 5-methylcytosine was determined by immunofluorescence. Results showed that at 18 h PI, mean H3K9ac intensity of vitrified embryos (11.8; n = 6) was higher than that of corresponding nonvitrified (fresh) controls (4.5; n = 6) and the fresh (3.2; n = 11) and vitrified (0.6; n = 7) 40-h groups (2-way ANOVA; P < 0.05). H3K9me2 in the fresh (36.9) and vitrified (32.5) 18-h embryos was similar but increased relative to both fresh (10.7) and vitrified (9.2) 40-h groups (P < 0.05). Mean DNA methylation (5MeC) in the fresh (31.6; n = 1) and vitrified (24.7; n = 3) 18-h groups was similar to that of the fresh 40-h group (19.8; n = 4) but higher than that of the vitrified 40-h group (15.0; n = 5; P < 0.05). To assess expression of POU5F1 and Nanog, qRT-PCR was performed on Day 8 blastocysts. Relative to controls (n = 9), mean POU5F1 and Nanog levels in vitrified blastocysts (n = 24) were 1.38- and 1.98-fold higher, respectively (one-way ANOVA; P > 0.05). In terms of in vitro development, Day 2 cleavage of vitrified zygotes (59%; n = 508) was similar to that of controls (66%; n = 340), but Day 8 blastocyst formation was reduced (9 v. 31%; t-test; P < 0.05). In vivo viability was assessed by oviducal transfer of 41 Day 1 embryos into 2 recipients. One pregnancy was established (50%), with 3 live kittens weighing 70, 79, and 131 g delivered without assistance on Day 65 of gestation. The 2 smaller kittens died within a few hours of birth, with the smallest exhibiting an umbilical hernia and organ exteriorization. The third kitten developed into a normal, healthy adult. In summary, mean H3K9me2, 5MeC, and POU5F1/Nanog expression of vitrified zygotes was similar to corresponding controls. H3K9ac increased at 18h PI as a result of vitrification, but was reduced after culture to 40 h PI. Although vitrified zygotes cleaved in vitro at rates similar to controls, blastocyst development was reduced. In vivo viability was demonstrated; however, postnatal survival of kittens produced was low.

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