56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 121 ◽  
Author(s):  
Y. M. Toishibekov ◽  
R. K. Tursunova ◽  
M. Sh. Yermekova
Keyword(s):  
Polar Body ◽  
Control Group ◽  
Polar Bodies ◽  
Maturation Medium ◽  
Chi Squared ◽  
Fetal Bovine ◽  
Second Polar Body ◽  
Cryopreservation Techniques ◽  
Humidified Air

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

41 THE EFFECT OF ULTRARAPID VITRIFICATION FOR SHEEP OOCYTE VIABILITY

2017 ◽  
Vol 29 (1) ◽  
pp. 128
Author(s):  
M. Toishibekov ◽  
Y. Toishibekov ◽  
M. Yermekova
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Polar Body ◽  
Control Group ◽  
Polar Bodies ◽  
Chi Squared ◽  
Fetal Bovine ◽  
Second Polar Body ◽  
Cryopreservation Techniques

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, have stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal’s body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). Ovaries from 19 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% fetal bovine serum, 5 μg mL−1 FSH, 5 μg mL−1 LH, 1 μg mL−1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then, oocytes were placed for 10 min in medium with Hoechst 33342 (3 μg mL−1) and cytochalasin B (7 μg mL−1) to facilitate enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into a cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation, the oocytes were activated for extrusion of the second polar body in 1 mg mL−1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL−1). After activation, the MPII were washed and cultured for 20 h. The control group received the same treatment but were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed that expulsion of the second polar body after activation was observed in more than 59.7% of the MPII that were not vitrified (control group), whereas 37.7% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

scholarly journals Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi

2021 ◽  
Vol 10 (2) ◽  
pp. 46
Author(s):  
Sepvian Dewi Kurniawati ◽  
Suryanie Sarudji ◽  
Widjiati Widjiati
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Petri Dish ◽  
Control Group ◽  
Maturation Medium ◽  
First Polar Body ◽  
Maturation Rate ◽  
Follicle Aspiration

This study was aimed to determine the effect of urea in maturation medium on in vitro oocyte maturation rate. The medium used was TCM-199 added with Hepes, NaHCO3, Kanamycin 0.15 IU/mL, PMSG, 0.15 IU/mL hCG, and 10% FBS. Cumulus oocyte complexes (COCs) of cows derived from follicle aspiration were divided into three groups. In control group (P0), the COCs were matured in vitro in a maturation medium without urea addition, meanwhile in the P1 and P2 groups, the medium was added with urea 20 and 40 mg/dL, respectively. Each petri dish contained three drops of maturation medium (300 µl/drops) according to the groups. Microdrops were coated with mineral oil and then incubated in a 5% CO2 incubator, at 39 ˚C with maximum humidity. Aceto-orcein staining was conducted to evaluate the maturation of oocytes based on the achievement of metaphase II phase that is indicated by the presence of metaphase plate and/or first polar body. The result showed that the oocyte maturation rates of P0, P1, and P2 were 51.25, 52.43 (p >0.05), and 46.88 % (p <0.05) respectively. It could be concluded that the presence of urea at 40 mg/dL in maturation medium reduced the percentage of bovine oocyte maturation in vitro.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P &lt; 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P &lt; 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P &lt; 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P &lt; 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P &lt; 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

6 EQUINE SPERM INDUCES PRONUCLEAR FORMATION BY INTRACYTOPLASMIC SPERM INJECTION IN BOVINE, SWINE, AND FELINE OOCYTES INDEPENDENTLY OF CHEMICAL ACTIVATION ASSISTANCE

2015 ◽  
Vol 27 (1) ◽  
pp. 95
Author(s):  
M. B. Rodríguez ◽  
A. Gambini ◽  
R. J. Bevacqua ◽  
D. F. Salamone
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Electric Pulse ◽  
Polar Body ◽  
Chemical Activation ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Chi Squared ◽  
Second Polar Body ◽  
Pronuclear Formation

Interspecific intracytoplasmic sperm injection (ICSI) is a valuable tool to study early events of fertilization in species for which oocyte availability is reduced. Equine in vitro fertilization remains unsuccessful and ICSI is the technique of choice for the in vitro production of high-value embryos. Therefore, the objective of this study was to evaluate the rate of pronuclear (PN) formation after ICSI with stallion sperm in bovine, swine and feline oocytes with or without chemical activation assistance. Ovaries from cows and pigs were collected at abattoirs whereas gonads from female domestic cats were obtained from ovariectomized animals at veterinary sterilization centers. Cumulus-oocyte complexes were matured in TCM-199 supplemented following standard protocols for each species. ICSI was performed in 100-μL drops of TALP-HEPES, using frozen-thawed semen from one stallion. Spermatozoa were held separate in 3-μL droplets of 7% (vol/vol) polyvinylpyrrolidone, where one of them was immobilized by swiping the injection pipette across its tail, and then injected into the matured oocyte. After ICSI, some oocytes were chemically activated with 5 μM ionomycin for 4 min (cow and cat) or with an electric pulse (sow) followed by 3 h in culture medium to allow extrusion of the second polar body and then exposure to 1.9 mM 6-DMAP solution for 3 h. Embryos were cultured in SOF medium. After 17 h of culture, embryos were stained with propidium iodide to identify the percentage of oocytes activated and with PN. Haploid and diploid parthenogenetic controls were included. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of the partenogenetic control groups were assessed. There were no statistical differences (chi-squared analysis) in PN formation between the activated and nonactivated groups within species. When the activated group was compared between the different species, no differences were observed. However, for the nonactivated group, significant differences were observed between species. The feline oocyte showed the higher percentage of PN and activation, whereas the bovine oocyte exhibited the lower rate of PN formation (cat: 22/27, 81.48%; swine: 19/39, 71.64%; cow:18/63, 43.07%). Our results suggest that the feline oocyte can be used as model to study fertilization events associated with the stallion sperm due to the higher efficiency in supporting PN formation. Our results indicate that the equine sperm is capable of inducing PN formation in these 3 species without further chemical activation assistance.

301 INTRACYTOPLASMIC SPERM INJECTION (ICSI) MEDIATED GENE TRANSFER ASSISTED BY ACTIVATION WITH A DOUBLE EXPOSURE TO IONOMYCIN AND 6-DIMETHYLAMINOPURINE OR DEHYDROLEUCODINE

2009 ◽  
Vol 21 (1) ◽  
pp. 247
Author(s):  
R. J. Bevacqua ◽  
F. Pereyra-Bonnet ◽  
R. Olivera ◽  
D. F. Salamone
Keyword(s):  
Gene Transfer ◽  
Embryo Development ◽  
Polar Body ◽  
Chemical Activation ◽  
Egfp Expression ◽  
Sperm Factor ◽  
Second Polar Body ◽  
Humidified Air ◽  
Novel Drug

ICSI-mediated gene transfer is a powerful technique used to produce transgenic mice and pigs. However, this method of transgenesis has not been applied in bovine due to low embryo development, which is presumed to be a consequence of a failure in sperm factor delivery after ICSI in this species. To bypass this problem, we assisted ICSI with chemical activation, employing two Ionomycin (Io) exposures and 6-Dimethylaminopurine (DMAP) or a novel drug, Dehydroleucodine (DhL). Cumulus–oocyte complexes were aspirated from ovaries obtained from a local slaughterhouse and in vitro matured in bicarbonate-buffered TCM-199 containing 10% FBS, 10 μg mL–1 FSH, 0.3 mm sodium pyruvate, 100 μm cysteamine and 10 UI mL–1 penicillin. IVM conditions were 6% CO2 in humidified air at 39°C for 24 h. MII oocytes were selected and used immediately for ICSI. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construction (pCX-EGFP) was carried out in Na citrate 2.8%, with 0.5 μg plasmid million–1 spermatozoa for 5 min at 0°C. Then, spermatozoa were used for ICSI. Injected oocytes were activated in 5 μm Io for 4 min and placed in TCM-199 for 3 h to allow second polar body emission. Afterwards, some of the oocytes were subjected to a second exposure of Io. Oocytes exposed once or twice to Io were then incubated with 2 mm DMAP (groups Io-DMAP and 2Io-DMAP) or 5 mm DhL (groups Io-DhL and 2Io-DhL) for 3 h. Control groups (Io and 2Io) were not treated with DMAP or DhL. Embryos were cultured in the IVM droplets. EGFP expression was daily evaluated in fluorescence microscope under blue light (488 nm). Significant differences between groups were evaluated by Fisher test (Table 1). DhL chemical activation improved neither development nor transgenesis rates. The double Io exposure significantly improved embryo development. The second exposure to Io previous chemical activation with DMAP resulted in an increase in the percentage of EGFP-expressing embryos. Our results indicate that activation with double Io-DMAP could be considered an alternative assistance for ICSI mediated gene transfer in bovine. Table 1.Effect of activation assisting transgenic ICSI on development and expression of bovine embryos

177 Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 213
Author(s):  
J. Keim ◽  
Y. Liu ◽  
I. Polejaeva
Keyword(s):  
Growth Factor ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Control Group ◽  
Control Medium ◽  
Maturation Medium ◽  
First Polar Body ◽  
Blastocyst Rate

In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P&lt;0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P&gt;0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P&lt;0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate. Supported by RFBR (18-29-07089) and UAES (1343).

205 HOLDING PIG OOCYTES AT 24°C PRIOR TO IN VITRO MATURATION ALTERS THE DEVELOPMENTAL CAPACITY AFTER IN VITRO FERTILISATION BUT NOT PARTHENOGENETIC ACTIVATION

2016 ◽  
Vol 28 (2) ◽  
pp. 233
Author(s):  
C. Quadalti ◽  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Polar Body ◽  
Cumulus Cells ◽  
Cell Number ◽  
T Test ◽  
Control Group ◽  
Parthenogenetic Activation ◽  
Significant Difference ◽  
Chi Squared ◽  
Developmental Capacity

The in vitro production of porcine embryos is of great interest because of the increasing importance of the swine as an animal model and a tissue donor for biomedical or biotechnological applications. Availability of ovaries at selected time of the day can be a limitation; therefore, the possibility to maintain immature oocytes for some hours can be very useful. The aim of this study is to determine whether holding recovered oocytes at 24°C for 24 h alters the maturation process and/or the developmental capacity. Immature sow oocytes were either matured in vitro for 42 h at 38.5°C (control group; CTR) or kept in 2 mL of HEPES-SOF in the dark at 24°C for 24 h before maturation (experimental group; +24 h). After maturation, cumulus cells were removed, and the number at metaphase II were recorded. For parthenogenetic activation (PGA), oocytes with a visible polar body were activated at 48 h of maturation as previously described (Lagutina et al. 2006). For IVF experiments frozen-thawed boar semen was prepared through a discontinuous density gradient, washed in TALP Ca2+-free, diluted in TALP : SOF = 1 : 1 supplemented with 6 mg mL–1 of fatty acid-free BSA, hypotaurine and epinephrine, mixed with oocytes after partial removal of the cumulus cells, at 43 h of maturation and cultured in 5% CO2 in humidified air at 38.5°C. After 24 h of IVF, oocytes were denuded and cultured in mSOF-1 in atmosphere of 5% O2 and 5% CO2. The same culture conditions were used after parthenogenetic activation. Half of the medium was changed with mSOF-1 at Day 3 and with mSOF-2 at Day 5. The cleavage and the cumulative Day 7 blastocyst (BLD7) rates and cell number of IVF BLD6 were recorded. For each group, blastocysts on Day 6 were fixed and cell number counted, whereas the other embryos were left in culture until Day 7 (cumulative D7 = BLD6 fixed + BLD7). All experiments were done in 3 replicates. The data were compared by Student’s t-test and chi-square test. Maturation rates as recorded for the presence of the polar body did not differ (CTR: 255/312, 82%; +24 h: 208/256, 81%). There was no significant difference (P < 0.05, chi-squared test) between CTR and +24 h group cleavage (144/165: 87% and 127/138: 92%, respectively) and BLD7 rate (47/165: 28% and 34/138: 25%, respectively) in the PGA. Whereas no difference (P < 0.05, chi-squared test) was observed between CTR and +24 h group cleavage (111/180: 62% and 99/186: 53%, respectively) in the IVF, but the BLD7 rate in +24 h group was significantly lower (48/180: 27% in the CTR group, 27/186: 15% in the +24 h group). However, the cell number of IVF BLD6 was not altered by holding at 24°C (n = 22: 25 ± 10 cells in the CTR, n = 8: 22 ± 13 cells in the +24 h) (P < 0.05, 2-tailed Student t-test). These experiments show that holding at 24°C for 24 h before maturation can alter the developmental capacity of IVF-produced embryos but not that of parthenogenetically activated ones. More replicates are needed to study the kinetics of maturation and to confirm our results. MitCare project (ERC n 322424) is acknowledged for support of this project.

Do embryonic polar bodies commit suicide?

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Dušan Fabian ◽  
Štefan Čikoš ◽  
Pavol Rehák ◽  
Juraj Koppel
Keyword(s):  
Polar Body ◽  
Point Of View ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Polar Bodies ◽  
First Polar Body ◽  
External Conditions ◽  
Living Organisms ◽  
Second Polar Body

SummaryThe extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

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.

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