scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Yongjin Lee ◽  
Hanna Lee ◽  
Joohyeong Lee ◽  
Seung Tae Lee ◽  
Geun-Shik Lee ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Glucose Supplementation

Summary This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

45 EFFECT OF TRICHOSTATIN A TREATMENT ON THE TERM DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER RABBIT EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103 ◽  
Author(s):  
Q. Meng ◽  
Z. Polgar ◽  
J. Liu ◽  
A. Dinnyes
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cumulus Cell ◽  
Cell Number ◽  
Pregnant Female ◽  
Rabbit Embryos

The efficiency of somatic cell nuclear transfer (SCNT) is low in the rabbit. So far, there have been few live births reported and most clones died within the first 3 weeks after birth. It has been shown that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, improved cloning efficiency in cattle (Enright et al. 2003 Biol. Reprod. 69, 896–901) and mice (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). Although a recent report indicated that TSA treatment could increase the cell number of rabbit SCNT blastocysts (Xu et al. 2007 Reprod. Fertil. Dev. 19, 165), term development of TSA-treated cloned embryos in this species has not been reported. In this study we investigated the effect of TSA treatment on the term development of somatic cell nuclear transfer (SCNT) rabbit embryos. The oocytes and cumulus cells were collected from superovulated Hycole hybrid rabbits. After staining with Hoechst 33342 and locating following 1–2 s of UV illumination, the nuclei of oocytes were removed by micromanipulation, A cumulus cell was then inserted into the perivitelline space and fused with the cytoplast with three 20 μs 3.2 kV cm–1 DC pulses. Fused embryos were activated using the same electrical parameters 1 h later, treated with 2 mm 6-dimethylaminopurine and 5 μg mL–1 cycloheximide for 1 h, and then subsequently cultured in Earles Balanced Salt Solution (EBSS) with or without 5 nm TSA for 10 h. The embryos were then cultured in EBSS either overnight (before ET) or for 4.5 days. Embryos were transferred at the 2- to 4-cell stages to the recipients 22 h after collection of the oocytes from the donors. Caesarean sections were performed on Day 30 post-ET. In vitro developmental data (Table 1) showed no differences in the cleavage, blastocyst rates, and blastocyst cell numbers between the TSA-treated or untreated cloned embryos. After ET in the TSA group, one pregnant female delivered 7 live and 3 stillborn pups, but all of the live pups died within 1 h to 19 days later. In the untreated group, one pregnant female gave birth to 2 live and 1 stillborn pup. One pup died within 1 h after birth; the other survived (2.5 months old when this abstract was submitted). In conclusion, the results indicate that TSA treatment has a limited effect on in vitro development of SCNT embryos, and both TSA-treated and untreated SCNT clones can develop to term in rabbit. The effects of TSA treatment on the health of clones need further investigation. Table 1. In vitro and in vivo development of SCNT rabbit embryos with or without TSA treatment This study was supported by Wellcome Trust (Grant No. 070246), EU FP6 (MEXT-CT-2003-509582, MRTN-CT-2006-035468), and Chinese-Hungarian Bilateral projects (TET CHN-28/04, CHN-41/05).

93 EFFECT OF SEASON ON QUALITY OF OOCYTES, RESULTS OF IN VITRO MATURATION, AND SOMATIC CELL NUCLEAR TRANSFER IN SWAMP BUFFALO

2007 ◽  
Vol 19 (1) ◽  
pp. 163
Author(s):  
N. T. Uoc ◽  
F. de Rennis ◽  
N. H. Duc ◽  
L. C. Bui ◽  
N. V. Hanh ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
Reproductive Activity ◽  
Blastocyst Stage ◽  
Swamp Buffalo

Reproductive activity in swamp buffalo is characterized by a clearly demonstrated anestrus season. The aim of the present study was to evaluate season effect on the oocyte collection, in vitro maturation, and somatic cell nuclear transfer. The ovaries collected from a slaughterhouse were divided into 3 groups according to the collection period: (1) G1: from January to April; G2: from May to August, which is characterized by higher climate temperature and low reproductive activity; and G3: from September to December. Cumulus–oocyte complexes (COCs) were aspirated from follicles 2-6 mm in diameter using an 18-gauge needle, washed in HEPES-buffered TCM-199 (Sigma-Aldrich, St Louis, MO, USA), and classified following 3 different quality levels: A (with 4–6 layers of cumulus cells), B (with 2–3 layers of cumulus cells), and C (few or without cumulus cells). The oocytes of A and B categories were used for IVM in maturation media currently used in cattle (TCM-199 medium + 10% fetal bovine serum) with an increase of FSH concentration (30 �g mL-1) and estradiol-17β (3 �g mL-1). Maturation was carried out at 39�C in a water-saturated incubator, under 5% CO2 for 22 h. The oocytes were observed for the cumulus expanding and the presence of polar body (PB). The oocytes with PB were used for further enucleation and cell nuclear transfer using buffalo quiescent fibroblast cells and the technique described previously (Nguyen et al. 2000 Theriogenology 53, 235). The percentages of intact and fused oocytes as well as reconstructed embryos developed to blastocyst stage were compared for the oocytes from G1 and G2. The results indicated that the average number of good quality COCs collected per ovary for the G1, G2, and G3 period were 6.00 � 4.08 (n = 426), 2.93 � 2.55 (n = 346), and 4.78 � 1.05 (n = 445), respectively. The percentages of A and B oocytes were 62.4% (1.58 � 0.51 vs. 2.17 � 1.54), 63.2% (0.90 � 0.32 vs. 0.95 � 0.50), and 54.7% (1.12 � 0.25 vs. 1.49 � 0.53), respectively; the maturation rate was 55.08%, 56.28%, and 52.16%, respectively. There were no significant differences between G1 and G2 in the percentage of intact and fused oocytes (93.7% and 59% for G1; 100% and 60% for G2, respectively), but the rate of embryos developed to blastocyst stage was higher for oocytes from G1 (18.5% vs. 10.2%). In conclusion, in swamp buffalo, the hot season affected significantly the number of oocytes collected per animal and the subsequent results of somatic cell nuclear transfer. The optimal period for working with buffalo oocyte is from January to April. This work was aupported by a grant from the Vietnam-Italy 3AB3 Project.

46 THREE STAGES ESTABLISHMENT OF IN VITRO MATURATION OF PORCINE OOCYTES USED AS RECIPIENT CYTOPLASTS FOR SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 129
Author(s):  
M. R. Lee ◽  
S. H. Park ◽  
T. S. Kim ◽  
S. Y. Kim ◽  
H. J. Eun ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Meiotic Division ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Protein Synthesis Inhibitor ◽  
Nuclear Reprogramming ◽  
In Vitro Oocyte Maturation

Oocytes at either anaphase/telophase of the first meiotic division (AI/TI) or metaphase of the second meiotic division (MII) are potential candidates used as recipient cytoplasts for somatic cell nuclear transfer (SCNT) because they contain active maturation-promoting factor (MPF), which causes nuclear membrane breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and may be essential for nuclear reprogramming (Campbell and Alberio 2003 Reprod. Suppl. 61, 477–494). In vitro maturation of porcine oocytes spends longer times (44 to 48 h), progresses asynchronously from immature stage and presents insufficient MPF for NEBD and PCC at AI/TI or MII. Here we have developed 3 stages establishment of in vitro oocyte maturation to select good quality of porcine oocytes used as recipient cytoplasts for SCNT. First, porcine oocytes were assessed by the activity of glucose-6-phosphate dehydrogenase (G6PD) before in vitro oocyte maturation. These oocytes with loss of expression of G6PD failed to enzymatically break down the dye, brilliant cresyl blue (BCB) and thus stain positively (Roca et al. 1998 Reprod. Fertil. Dev. 10, 479–485). Positively BCB stained oocytes reached to MII (67.0% v. 58.4%) and produced more parthenogenetic blastocysts (50.2% v. 29.6%) in comparison with negatively BCB stained oocytes (P < 0.05). Second, positively BCB stained oocytes were treated with 5 μg mL–1 cycloheximide (CHXM, a nonspecific protein-synthesis inhibitor) to block meiotic progression for synchronizing the cell cycle of oocytes for 16 h. All of the oocytes (130/130) were efficiently synchronized at the germinal vesicle (GV) stage by treatment with CHXM, whereas control oocytes (w/o CHXM) were passed through GV stage (80/176). Retrieval from treatment of CHXM could reversibly induce meiotic resumption and progresses synchronously in porcine oocytes. Following incubation with gonadotropin hormones [epidermal growth factor (EGF) 10 ng mL–1, LH 5μg mL–1, FSH 1 μg mL–1], treatment with 5 mM caffeine (a phosphatase inhibitor) for 12 h significantly increased the activity of MPF in porcine oocytes (P < 0.05). However, there was no difference between CHXM treated and nontreated group (21.9% v. 22.3%) in the developmental rate to the blastocyst stage of parthenogenetic embryos. At least 3 replicates were performed. These data may contribute to an improved nuclear reprogramming in SCNT.

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