265 INHIBITION OF CLASS III PHOSPHATIDYLINOSITOL-3-KINASE, BY 3-METHYLADENINE, REVERSIBLY ARRESTS PORCINE OOCYTES AT GERMINAL VESICLE STAGE

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
M. R. Park ◽  
M. K. Gupta ◽  
S. J. Uhm ◽  
S. T. Shin ◽  
Y. M. Han ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Indirect Evidence ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Class Iii ◽  
Cleavage Rate ◽  
Chi Square ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P < 0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2 ± 0.8 v. 54.0 ± 10.1%) or absence (96.5 ± 1.8 v. 41.0 ± 17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached metaphase II stage at 42 h of in vitro maturation and did not differ (P > 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage. This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.

70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

2006 ◽  
Vol 18 (2) ◽  
pp. 143
Author(s):  
D. Salamone ◽  
M. Catala ◽  
A. Gibbons ◽  
F. Pereyra Bonnet ◽  
M. Cueto
Keyword(s):  
Granulosa Cells ◽  
Room Temperature ◽  
Uv Light ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
First Passage ◽  
Chi Square ◽  
Donor Nucleus ◽  
Chi Square Test

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts. Table 1. Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

2011 ◽  
Vol 75 (5) ◽  
pp. 940-950 ◽  
Author(s):  
Myung Rae Park ◽  
Mukesh Kumar Gupta ◽  
Hye Ran Lee ◽  
Ziban Chandra Das ◽  
Sang Jun Uhm ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
W. Huanca ◽  
R. Condori ◽  
J. Cainzos ◽  
M. Chileno ◽  
L. Quintela ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hypodermic Needle ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Time ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Maturation Medium

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.

Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes

2003 ◽  
Vol 179 (1) ◽  
pp. 25-34 ◽  
Author(s):  
M Shimada ◽  
J Ito ◽  
Y Yamashita ◽  
T Okazaki ◽  
N Isobe
Keyword(s):  
High Activity ◽  
Cell Function ◽  
Kinase Inhibitor ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Phosphatidylinositol 3 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Phosphatidylinositol 3

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

246 INDUCTION OF MEIOTIC ARREST IN IMMATURE FELINE OOCYTES WITH ROSCOVITINE AND DIBUTYRYL CYCLIC-AMP

2009 ◽  
Vol 21 (1) ◽  
pp. 221
Author(s):  
C. B. Hanna ◽  
T. M. Glover ◽  
C. R. Long
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Control Culture ◽  
Basic Medium ◽  
Meiotic Arrest ◽  
Chi Square ◽  
Exact Test ◽  
Chromatin Configuration

Successful application of some assisted reproduction technologies in feline species requires the use of competent, meiotically mature ova, which can be difficult to obtain in sufficient quantities. Meiotic arrest of immature feline oocytes would allow the accumulation of oocytes over time to maximize laboratory resources. Two meiosis inhibitors commonly used in other species, roscovitine (ROS) and dbcAMP, were evaluated for the ability to maintain a germinal vesicle (GV) in immature feline oocytes after 24 h of in vitro culture. Feline ovaries were obtained from routine ovariohysterectomies and oocytes with homogenous cytoplasm and at least 2 layers of cumulus cells were selected. All oocytes were cultured in a basic medium modified from Gomez et al. (2000 Reprod. Fertil. Dev.) consisting of TCM-199 with Earle’s salts, 0.3% fraction V bovine serum albumin, 2.0 mm L-glutamine, 1.12 mm L-cysteine, 2.2 mm calcium lactate, 0.36 mm sodium pyruvate, 100 μm cysteamine, 10 ng μL–1 epidermal growth factor, 1 μg mL–1 estradiol, and 50 μg mL–1 gentamicin, with or without meiotic inhibitor. After 24 h of culture, cumulus cells were removed; oocytes were fixed, permeabilized, and stained with 5 μg mL–1 of Hoechst 33342; and chromatin configuration was assessed under ultraviolet fluorescence. In 4 replicates of Experiment 1, oocytes were cultured with either 25 nm ROS, 1 mm dbcAMP, both ROS and dbcAMP (Both), or without inhibitor (Control). A greater proportion of oocytes retained the GV when cultured with Both compared with ROS, dbcAMP, or Control (90.6, 67.8, 25.0, and 14.8%, respectively; chi-square P < 0.05), and ROS alone was superior to dbcAMP or Control. Culture of oocytes in the base medium plus 0.5 IU mL–1 eCG and 1.0 IU mL–1 hCG after arrest showed that pretreatment with Both did not decrease their ability to resume meiosis (87.9 v. 87.0%, respectively). Given the low percentage of oocytes with a retained GV in the presence of 1.0 mm dbcAMP, Experiment 2 evaluated the concentration of dbcAMP required to inhibit meiosis. Oocytes were cultured over 4 replicates in the base media containing 0, 0.1, 0.5, 1.0, 2.0, and 10.0 mm dbcAMP. After 24 h of culture, a greater number of oocytes were arrested at the GV stage when cultured in 10 mM dbcAMP (39.8%) than in 0, 0.1, 1.0, and 2.0 mM dbcAMP (22.0, 20.4, 25.8, and 26.3%, respectively; Fisher’s exact test P < 0.05), but there was no difference from those cultured in 0.5 mm dbcAMP (28.1%). For many species, 1.0 mm dbcAMP is commonly used to inhibit meiosis successfully for 24 h. However, dbcAMP alone did not effectively arrest meiosis, but when combined with ROS, it tended to improve meiotic arrest. Experiment 2 indicates that at least 10.0 mm dbcAMP is required to show a significant effect of meiotic inhibition in feline oocytes. Under these culture conditions, dbcAMP at levels up to 10 mm were not effective at inducing meiotic arrest. Interestingly, ROS and dbcAMP may act synergistically to successfully induce a reversible inhibition of meiosis in a high percentage of feline oocytes.

scholarly journals AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes

Reproduction ◽  
2009 ◽  
Vol 138 (4) ◽  
pp. 645-654 ◽  
Author(s):  
Jaroslav Kalous ◽  
Michal Kubelka ◽  
Petr Šolc ◽  
Andrej Šušor ◽  
Jan Motlík
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinase B ◽  
Specific Inhibitor ◽  
Germinal Vesicle ◽  
Akt Phosphorylation ◽  
Meiotic Progression ◽  
Significant Acceleration ◽  
Protein Kinase Akt

The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) maturedin vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 μM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

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.

Sign in / Sign up

Export Citation Format

Share Document