scholarly journals Histone demethylase KDM4A overexpression improved the efficiency of corrected human tripronuclear zygote development

2021 ◽  
Author(s):  
Hai-Ying Zhu ◽  
Xiang-Jin Kang ◽  
Long Jin ◽  
Pu-Yao Zhang ◽  
Han Wu ◽  
...  
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Lysine Specific Demethylase ◽  
Zygote Development ◽  
H3k9me3 Level ◽  
Genome Activation ◽  
Formation Efficiency ◽  
Human Zygotes

Abstract Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, 8-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000 ng/µl lysine-specific demethylase 4 A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.

113 EFFECT OF PLANT PROTEIN ON DEVELOPMENT AND QUALITY OF CULTURED IN VITRO PORCINE ZYGOTES

2009 ◽  
Vol 21 (1) ◽  
pp. 157 ◽  
Author(s):  
B. Gajda ◽  
I. Grad ◽  
Z. Smorag
Keyword(s):  
Serum Albumin ◽  
Culture Media ◽  
Quality Criteria ◽  
Cell Number ◽  
Developmental Competence ◽  
Plant Protein ◽  
Control Group ◽  
Group 1

Basic culture media are usually supplemented with serum albumin or serum, which contain amino acids that play an important role as energy sources, osmoregulators, and pH stabilizers. However, the presence of undefined serum in culture media introduces a variation from batch to batch and increases viral or prion contamination risk. The aim of the present study was to investigate the possibility of using plant protein substitute (PP) in place of bovine serum albumin (BSA) during in vitro culture of porcine zygotes. The PP is a mixture of several plant proteins and soya lecithin prepared using a high pressure homogenization process. The experiment was done on pig zygotes obtained surgically from superovulated gilts at 24–26 h after insemination. Morphologically normal zygotes were cultured in vitro in 5% CO2 in air at 39° in NCSU-23 medium supplemented with: 0.002 g mL–1 (group 1), 0.004 g mL–1 (group 2), 0.008 g mL–1 (group 3) PP or 0.004 g mL–1 BSA (control group). Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL method. Results were analyzed by Chi-square test. There were no differences in cleavage rates on Day 2 between zygotes cultured in NCSU-23 medium supplemented with PP (86.0, 88.0, 84.8; group 1 to 3, respectively) and BSA (91.0%, control group). Culture with 0.008 g mL–1 PP increased morula (85.7%) and blastocyst (69.2%) production as compared with control (75.0% and 56.3%, respectively; P < 0.05) and 0.002 g mL–1 PP (79.5% and 51.8%, respectively; P < 0.05). The mean number of cells in Day 7 blastocysts cultured in NCSU-23 medium + 0.004 g mL–1 BSA was lower (P < 0.05) than in NCSU-23 + 0.004 g mL–1 PP (39.1 v. 43.7, respectively). The blastocysts cultured in NCSU-23 medium + 0.002 g mL–1 PP had higher average number of apoptotic nuclei (13.0) as compared with the control (6.5) and 0.004 g mL–1 PP (6.9). In conclusion, this study suggest the positive effect of PP on development in vitro of porcine zygotes to the morula/blastocyst stage. However, further studies are required to determine the quality of the embryos cultured with PP. This study was supported by Scientific Net of Animal Reproduction Biotechnology.

scholarly journals High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 347-355 ◽  
Author(s):  
Ikuko Yashiro ◽  
Miho Tagiri ◽  
Hayato Ogawa ◽  
Kazuya Tashima ◽  
Seiji Takashima ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Total Cell Number ◽  
Recovery Culture ◽  
Bovine Oocytes

The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

38 Improved Cloning Efficiency and Developmental Potential in Bovine Somatic Cell Nuclear Transfer with the New Technology

2018 ◽  
Vol 30 (1) ◽  
pp. 158
Author(s):  
L. Xu ◽  
M.-D. Joo ◽  
A. Mesalam ◽  
S.-H. Song ◽  
S. Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Reverse Transcription ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Methyl Transferase ◽  
Developmental Potential

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by IVF; however, its efficiency remains low. In this study, we examined the effects of cytoplasm restoration of enucleated oocyte, by injecting ~30% of the cytoplasm of a donor oocyte to restore the enucleated oocyte cytoplasm volume to normal, on the developmental competence and quality of bovine cloned embryos during pre-implantation using the TUNEL assay, quantitative reverse transcription PCR (RT-qPCR) and immunocytochemistry. The experiment was conducted in 6 replicates. The differences in embryo development and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (P < 0.05) in the cytoplasmic injected group than in the traditional SCNT group (61.5 ± 1.3% v. 39.7 ± 2.1% and 28.9 ± 0.8% v. 20.2 ± 1.3%, respectively). Furthermore, the beneficial effects of cytoplasmic injection on the cloned embryos were associated with a significantly increased (P < 0.05) total cell number in Day 8 blastocysts compared with the traditional SCNT group (176.2 ± 6.5 v. 119.3 ± 7.7; P < 0.05); however, there was no difference (P > 0.05) between the number of apoptotic cells per blastocyst in the cytoplasmic injected group and in the traditional SCNT group (3.5 ± 1.1 v. 4.1 ± 0.8). Moreover, cytoplasm restoration of enucleated oocyte significantly increased (P < 0.05) mitochondrial activity, as identified by MitoTracker Green (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription-qPCR showed that the mRNA levels of DNA methyl-transferase 1 and DNA methyl-transferase 3a were significantly decreased (P < 0.05) in cytoplasmic injected group compared with the traditional SCNT group, but did not significantly differ (P > 0.05) between the cytoplasmic injected and IVF groups. Taken together, these data suggest that cytoplasm restoration of enucleated oocyte improves in vitro developmental competence and quality of bovine cloned embryos, as evidenced by increased total cell numbers, reprogramming efficiency, and mitochondria activity. This work was partly supported by grant from the Next-Generation BioGreen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co. Felix Pets) and BK21plus.

332 EMBRYONIC DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER OF PORCINE OOCYTES MEIOTICALLY INHIBITED WITH ROSCOVITINE

2006 ◽  
Vol 18 (2) ◽  
pp. 273
Author(s):  
S. W. Kim ◽  
D. H. Kim ◽  
J. S. Seo ◽  
G. S. Im ◽  
B. C. Yang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Apoptotic Cell ◽  
Treated Group ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Maturation Medium

Numerous factors affect on the developmental competence of cloned embryos, and one of the factors might be the disturbed synchronization of nuclear and cytoplasm maturation. Roscovitine, a purine known to specifically inhibit M-phase promoting factor (MPF) kinase activity by blocking the ATP in numerous cell systems, has been successfully used in maintaining porcine oocytes at GV stage without affecting their developmental potential. However, developmental ability of roscovitine treated porcine oocytes after nuclear transfer has not been evaluated. The purpose of this study was to examine the development of nuclear transferred porcine embryos after meiotic inhibition with roscovitine (ROS). Cumulus-oocyte complexes (COCs) were collected from antral follicles of slaughtered prepubertal gilts. COCs were cultured in pre-maturation medium (TCM-199 containing 50 �M Roscovitine) for 24 h, and then further cultured in conventional maturation medium for 44 h. A control group was cultured in the maturation medium for 44 h. Matured oocytes were enucleated and a porcine fetus cell was inserted into each enucleated oocyte. Couplets were simultaneously fused and activated with electric pulse of two 1.2 kV/cm for 30 �s. Nuclear transferred (NT) embryos were cultured in PZM-1 medium for 6 days (five replicates). Apoptotic cell death was analyzed by using a TUNEL assay and total cell number was examined by Hoechest 33342 counterstaining. At 3 h after fusion, NT embryos were fixed for microfilament staining. Data were analyzed by ANOVA and Student's t-test. The rates of fusion, cleavage, and blastocyst formation of the ROS-treated group (85, 68, and 18%, respectively) after nuclear transfer did not differ from control (78, 76, and 16%, respectively). The cell number in blastocysts of the ROS-treated group (30.8 � 10.6) was significantly lower than that of the control (42.3 � 13.7) (P < 0.01), but the mean proportion of apoptotic cells was not different between the two groups (6.9 � 7.1 and 4.8 � 4.9% for control and ROS group, respectively). Recovery of microfilaments after fusion was delayed in NT embryos derived from ROS-treated oocytes. This study demonstrated that porcine oocytes pre-cultured for 24 h in presence of roscovitine can be developed to blastocysts after somatic cell nuclear transfer. This could provide flexibility for studying porcine oocyte development and embryo cloning.

147 QUALITY OF PORCINE EMBRYOS CULTURED WITH HYALURONAN

2010 ◽  
Vol 22 (1) ◽  
pp. 232
Author(s):  
B. Gajda ◽  
I. Grad ◽  
E. van der Tuin ◽  
Z. Smorag
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Apoptotic Index ◽  
Total Cell ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Group 2 ◽  
Group 1

Hyaluronan (HA) is a high molecular weight polysaccharide found in the mammalian follicular, oviduct, and uterine fluids. When HA is added in maturation and culture media, it improves the developmental potential of bovine (Stojkovic M et al. 2002 Reproduction 124, 141-153; Palasz AT et al. 2008 Zygote 16, 39-47), and porcine oocytes (Sato E et al. 1990 Mol. Reprod. Dev. 26, 391-397) and embryos (Miyano T et al. 1994 Theriogenology 41, 1299-1305). Physiological concentration of HA in follicular, oviductal, and uterine fluids of pigs range from 0.04 to 1.83 mg mL-1 (Kano K et al. 1998 Biol. Reprod. 58, 1226-1232). The aim of the present study was to investigate the effect of different concentrations of HA on the development and quality of cultured porcine embryos. Zygotes from superovulated pigs were cultured in vitro in NCSU-23 medium supplemented with BSA and 0 mg mL-1 (control group), 0.25 mg mL-1 (Exp. Group 1), and 0.5 mg mL-1 (Exp. Group 2) of HA (Animal Pharma BV). Experiments were replicated 3 times with 30 to 40 embryos per each treatment group. Embryos were cultured up to the blastocyst stage at 39°C in an atmosphere of 5% CO2 in air, in 4-well plastic dishes, which contained approximately 0.8 mL of the NCSU-23 medium. Embryo quality criteria were cleavage (on Day 2 after in vitro culture), morula (on Day 4) and blastocyst (on Days 6 to 8) rates, total cell number per blastocyst, and degree of apoptosis (on Day 7) assessed by TUNEL method. Results were analyzed by ANOVA test. There was no difference in percentage of cleaved embryos between control and treated Group 1 and 2.The proportion of embryos developed to the morula and blastocyst stage was 80.0 and 60.0% for Group 1 (0.25 mg of HA), 73.7 and 44.7% for Group 2 (0.5 mg of HA), and 73.4 and 46.7% for control, respectively (difference NS). Supplementation with HA did not increase the cell number of the blastocysts but significantly reduced number of apoptotic nuclei from 2.0 for control to 0.7 (P < 0.01) and 0.6 (P < 0.01) for Group 1 and 2, respectively, and apoptotic index from 9.70 for control to 3.01 (P < 0.05) and 1.95 (P < 0.05) for Group 1 and 2, respectively. These results indicate that supplementation of culture medium NCSU-23 with HA improves the quality (assessed by apoptotic index) of pig embryos but does not increase the total cell number in pig blastocysts as reported by Kim HS et al. 2005 (Theriogenology 63, 1167-1180). However, further research to test the HA’s effect on cryopreservation of in vitro and in vivo produced pig embryos are needed.

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1061 ◽  
Author(s):  
RD Schramm ◽  
BD Bavister
Keyword(s):  
Growth Hormone ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Morula Stage

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

scholarly journals 147ION COMPOSITION OF CULTURE MEDIUM INFLUENCES MITOCHONDRIAL DISTRIBUTION AND BLASTOCYST DEVELOPMENT OF PREIMPLANTATION PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
M.L. Conover-Sparman ◽  
R.L. Krisher
Keyword(s):  
Culture Medium ◽  
Krebs Cycle ◽  
Cell Number ◽  
Developmental Competence ◽  
Homogeneous Distribution ◽  
Distribution Data ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Mitochondrial Distribution

Elevated intracellular calcium (Ca2+) concentrations impair hamster embryo metabolism and viability (Lane M and Bavister B 1998 Biol. Reprod. 59, 1000–1007). Extracellular magnesium (Mg2+) regulates intracellular Ca2+ by controlling its uptake and release. In the present study, we examined the effects of altering Ca2+ and Mg2+ ion concentrations in Purdue Porcine Medium (PPM1) on porcine embryo mitochondrial distribution, metabolic (glycolytic and Krebs cycle) activity, and in vitro developmental potential. Cumulus-oocyte complexes collected from abattoir ovaries were matured for 40–42h, inseminated with 5×105 sperm mL−1 for 5h, and initially cultured in 1:0.4 or 2:1 ratio of Ca2+ to Mg2+ (concentrations in mM) at 38.7°C, in 6% CO2, 10% O2, balance N2. At 22–26, 46–50, and 70–74h post-insemination, 2-, 4-, and 8-cell embryos, respectively, were removed from culture to evaluate mitochondrial distribution (confocal microscopy after tetramethylrhodamine methyl ester staining) and glycolytic and Krebs cycle activity (5-[3H]-glucose and 2-[C14]-pyruvate, respectively). Remaining embryos were further cultured to determine developmental competence (2:1, n=548; 1:0.4, n=560). Cleavage was assessed on Day 3 (2:1, n=552; 1:0.4, n=560) of culture. All data were analyzed using GLM ANOVA, except mitochondrial distribution data which were analyzed using GLIMMIX. A majority (P&lt;0.05) of 2-cell (65%, 13/20) and 4-cell (67%, 22/33) embryos cultured in 2:1 displayed a homogeneous mitochondrial distribution. More (70%, 21/30; P&lt;0.05) 8-cell embryos cultured in 2:1 had a perinuclear mitochondrial distribution. When cultured in 1:0.4, a majority (61%, 14/23; P&lt;0.05) of 2-cell embryos displayed a cortical mitochondrial distribution, whereas most (P&lt;0.05) 4-cell (66%, 19/29) and 8-cell embryos (69%, 18/26) displayed a homogeneous distribution. Glycolytic and Krebs cycle activities were similar (P&gt;0.05) between treatments and across all cell stages examined. Treatment had no effect (P&gt;0.05) on cleavage or blastocyst total cell number. Unlike hamster embryos, culturing pig embryos in a higher Ca2+ concentration resulted in more embryos developing to the blastocyst stage. Culture medium containing 2mM Ca2+ and 1mMMg2+ best supports in vitro blastocyst development, possibly by supporting a more correct mitochondrial distribution. These results are not mediated via changes in glycolytic or Krebs cycle activity, thus suggesting that another cellular mechanism plays a key role in developmental competence in early pig embryos. Table 1 Effects of Ca2+:Mg2+ on porcine embryonic development and metabolic activity (mean±SEM).

scholarly journals Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus–oocyte complexes exposed to heat shock during in vitro maturation

Reproduction ◽  
2013 ◽  
Vol 146 (4) ◽  
pp. 407-417 ◽  
Author(s):  
A Z Balboula ◽  
K Yamanaka ◽  
M Sakatani ◽  
M Kawahara ◽  
A O Hegab ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cathepsin B ◽  
Caspase 3 ◽  
Developmental Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining

Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus–oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.

23 DEVELOPMENTAL POTENTIAL OF CLONED TRANSGENIC PORCINE EMBRYOS PRODUCED BY SERIAL NUCLEAR TRANSFER CAN BE IMPROVED BY TREATMENT WITH HISTONE DEACETYLASE INHIBITORS

2012 ◽  
Vol 24 (1) ◽  
pp. 123
Author(s):  
M. Kurome ◽  
V. Zakhartchenko ◽  
B. Kessler ◽  
T. Güngör ◽  
A. Richter ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Cell Number ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Significant Difference ◽  
Post Implantation

Recently, we generated cloned transgenic pigs by nuclear transfer (NT) using fetal fibroblasts transfected with a LEA29Y gene specifically expressed in pancreatic β-cells (INS-LEA). Transfer of 216 NT embryos into 3 recipients resulted in the birth of 9 piglets. Furthermore, we examined serial NT with donor cells of the INS-LEA cloned pigs as a means of propagating the genotype of these valuable animals. Surprisingly, no piglets were obtained after transfer of 512 NT embryos into 5 recipients, which might be due to epigenetic alterations that presumably occurred during post-implantation development of the first round cloned embryos or during nuclear reprogramming in the second round of NT. In this study we tested whether in vitro development of re-cloned embryos can be improved by their treatment with histone deacetylase inhibitors (HDACi), scriptaid and suberoylanilide hydroxamic acid (SAHA). As nuclear donors, ear fibroblast cells derived from the INS-LEA cloned pig were used. Nuclear transfer was performed using in vitro-matured oocytes as previously reported (Kurome et al. 2006, Transgenic Res. 15, 229–240). After activation, reconstructed embryos were treated immediately by scriptaid (500 nM) and SAHA (10 μM) for 16 and 10 h, respectively. Development of NT embryos was assessed by cleavage and blastocyst formation during culture for 7 days. The cell number of blastocysts was also counted after fixation and staining. There was no significant difference in the cleavage rate between treated and non-treated by both HDACi, whereas treatment of NT embryos with scriptaid or SAHA significantly enhanced their development to blastocyst compared with non-treated NT embryos (22.2%, 43/194 and 22.7%, 34/150 vs 7.7%, 15/195 and 12.3%, 18/146, respectively; P < 0.05). Notably, blastocyst rates obtained after treatment of re-cloned embryos with HDACi were similar to those in the first round of NT (21.2%, 33/156). Treatment of NT embryos with HDACi did not increase mean cell number of blastocysts compared with non-treated embryos. The results of our study show that in vitro developmental competence of embryos produced by serial NT can be improved by both HDACi used, scriptaid as well as SAHA, which has not been reported before in pig cloning. To determine the post-implantation developmental potential, re-cloned embryos treated with HDACi will be transferred to surrogate gilts. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung and Mukoviszidose e.V.

40 Administration of 0.1µM melatonin during invitro maturation of bovine oocytes regulates autophagy levels in produced embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 127
Author(s):  
M. El-Sheikh ◽  
A. A. Mesalam ◽  
K.-L. Lee ◽  
I.-K. Kong
Keyword(s):  
Cumulus Cells ◽  
Treated Group ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Dapi Staining ◽  
Melatonin Treatment ◽  
Developmental Potential ◽  
Bovine Oocytes

Melatonin, the antioxidant pineal hormone, is a strong regulator for various cellular processes essential for reproduction. Although the protective role of 0.1µM melatonin against the toxicity of different anti-developmental compounds has been elucidated in numerous studies, its effect on the autophagy level in invitro-produced blastocysts has not been entirely clarified. In this study, oocytes were incubated for 24h in the presence and absence of melatonin, administered during IVM, to investigate the effect of 0.1µM melatonin on the developmental competence of bovine oocytes and pre-implantation embryos, autophagy, and quality of embryos. The developmental potential of embryos were basically the stages from oocytes fertilization to blastocyst production. Gene expression levels were evaluated in matured oocytes, whereas blastocysts were used for immunofluorescence experiments. The differences between treated and control groups were analysed using Student’s t-test (GraphPad Prism version 6; GraphPad Inc.), where P-values &lt;0.05 were considered significant. Results showed that oocyte maturation, Day-4 total cleavage, and Day-8 blastocyst development rates were not significantly improved (melatonin: 72±2 vs. control: 69±2 for cleavage rate, and melatonin: 33±1 vs. control: 31±2 for control for Day-8 blastocyst; P&gt;0.05), whereas the level of reactive oxygen species (ROS) was reduced (P&lt;0.05) with addition of melatonin. Using RT-qPCR, cumulus cells-related (HAS2) and apoptosis-related (Bcl2 and SOD2) genes were upregulated, whereas BAX was downregulated in melatonin-treated oocytes. Using immunofluorescence, apoptosis (caspase-3) and autophagy (Beclin-1 and LC3) markers were underexpressed, whereas the PI3K survival protein (P&lt;0.05) and matrix metalloproteinases (MMP-2 and MMP-9; P&gt;0.05) were overexpressed, in Day-8 embryos of melatonin-treatment. Additionally, the total number of cells per blastocysts, inspected via nuclei-based 4′,6-diamidino-2-phenylindole (DAPI) staining was higher in the melatonin-treated group (P&lt;0.05). Taken together, our study demonstrates that 0.1µM melatonin treatment during IVM does not interfere with developmental competence, but improves the quality of IVF-produced embryos by lowering the incidence of autophagy.

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